Virus Assembly:
The assembly of Viral structural proteins and nucleic acid (Viral dna or Viral rna) to form a Virus particle.
JoVE Immunology and Infection
Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly
1Department of Plant Pathology and Microbiology, University of California, Riverside, 2Department of Bioengineering, University of California, Riverside
A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.
JoVE Immunology and Infection
Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles
Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.
JoVE General
Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
Molecular Oncology Research Institute, Tufts University
This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.
JoVE Immunology and Infection
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY
We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.
JoVE General
Lentivirus Production
Diabetes Center, University of California, San Francisco - UCSF
To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.
JoVE Immunology and Infection
Production and Titering of Recombinant Adeno-associated Viral Vectors
1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University
Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.
JoVE General
Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
JoVE Immunology and Infection
RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania
Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
JoVE Immunology and Infection
Retroviral Transduction of T-cell Receptors in Mouse T-cells
1NYU Cancer institute, New York University School of Medicine, 2Program in Structural Biology, New York University School of Medicine
We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction
JoVE Immunology and Infection
Visualizing Dengue Virus through Alexa Fluor Labeling
1Defence Medical and Environmental Research Institute, DSO National Laboratories, 2Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School
Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.
JoVE Immunology and Infection
Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ)
Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).
JoVE Immunology and Infection
In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus
Experimental Pathology, University of Texas Medical Branch
Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.
JoVE Immunology and Infection
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
Department of Immunology, Duke University
We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.
JoVE Neuroscience
Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System
In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.
JoVE Immunology and Infection
Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs
1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture
A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.
JoVE Immunology and Infection
Recombinant Retroviral Production and Infection of B Cells
1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 2Herbert Irving Comprehensive Cancer Center, Columbia University
An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.
JoVE Clinical and Translational Medicine
Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University, 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University
A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.
JoVE General
Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.
JoVE General
Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center
A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.
JoVE Immunology and Infection
Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein
Institute for Experimental Pathology, University of Iceland
We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.
JoVE Clinical and Translational Medicine
Modeling and Imaging 3-Dimensional Collective Cell Invasion
1Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 2The Beatson Institute for Cancer Research
Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.
JoVE Neuroscience
Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5
1Institute for Cell Engineering Neuroregeneration and Stem Cell Programs, Johns Hopkins University School of Medicine, 2Departments of Neurology, Neuroscience, and Oncology, Johns Hopkins University School of Medicine
In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.
JoVE Clinical and Translational Medicine
High Content Screening in Neurodegenerative Diseases
1Department of Clinical Genetics, VU University Medical Center, 2Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam
We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.
JoVE Immunology and Infection
Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University
We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.
JoVE Immunology and Infection
Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.
JoVE Immunology and Infection
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Department of Pathology, University of Texas Medical Branch
The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.
JoVE Clinical and Translational Medicine
High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors
Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida
In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.
JoVE Clinical and Translational Medicine
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine
We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.
JoVE Clinical and Translational Medicine
DNA Vector-based RNA Interference to Study Gene Function in Cancer
1Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, 2Department of Pathology and Comprehensive Cancer Center, Wake Forest University School of Medicine
RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.
JoVE General
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Department of Biological Sciences, The University of Memphis
Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.
JoVE General
Generation of Mice Derived from Induced Pluripotent Stem Cells
1Dorris Neuroscience Center & Department of Cell Biology, The Scripps Research Institute, 2Mouse Genetics Core Facility, The Scripps Research Institute
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
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
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
JoVE Immunology and Infection
Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory
This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.
JoVE Immunology and Infection
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.
JoVE Clinical and Translational Medicine
Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University, 3Omics Laboratory, University of Iowa, 4Department of Ophthalmology and Visual Sciences, University of Iowa
This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.
JoVE Neuroscience
Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations
Department of Experimental Neurobiology, The Neurosciences Institute
We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.
JoVE Immunology and Infection
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Department of Pharmacology, School of Medicine, New York University
The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.
JoVE Neuroscience
A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats
Psychology, University of Toronto Scarborough
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
JoVE General
Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method
Molecular Biophysics and Biochemistry, Yale University
This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).
JoVE Immunology and Infection
Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells
Department of Virology, Universitätsklinikum Freiburg
Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.
JoVE Immunology and Infection
Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology
The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.
JoVE Immunology and Infection
Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
1Research Group Immune Regulation, Helmholtz Centre for Infection Research, 2Research Group Infection Immunology, Institute of Medical Microbiology, Otto-von-Guericke University, 3Department of Experimental Immunology, Helmholtz Centre for Infection Research
We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.
JoVE Immunology and Infection
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Viral Populations and Pathogenesis lab and CNRS 3015, Institut Pasteur
The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.
JoVE Immunology and Infection
Estimating Virus Production Rates in Aquatic Systems
Department of Microbiology, University of Tennessee
The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.
JoVE Neuroscience
Intracranial Injection of Adeno-associated Viral Vectors
Neurobiology and Anatomy, University of Rochester
Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.
JoVE Immunology and Infection
Generation of Recombinant Influenza Virus from Plasmid DNA
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.
JoVE Immunology and Infection
Particle Agglutination Method for Poliovirus Identification
1Department of Virology II, National Institute of Infectious Diseases, 2New Product Design Department, Fujirebio Inc.
A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.
JoVE Immunology and Infection
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
JoVE Application Notes
CryoStor Cryopreservation Protocol - ADVERTISEMENT
BioLife Solutions, Inc.
CryoStor cryopreservation solutions are used to prepare and preserve cells in ultra low temperature environments, without the need for serum, proteins, or high levels of cytotoxic agents.
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