Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering.
In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20%) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100%) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.
23 Related JoVE Articles!
Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research
Institutions: Karlsruhe Institute of Technology.
A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.
Cellular Biology, Issue 15, Interview, bioreactors, cell culture systems, 3D cell culture, stem cells
Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology
Institutions: Rensselaer Polytechnic Institute, Stemorgan Inc., Technical University of Munich, Wuhan University, The Scripps Research Institute, Tokyo University of Science.
Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.
Bioengineering, Issue 88, cartilage, inkjet printing, chondrocytes, hydrogel, photopolymerization, tissue engineering
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Institutions: National Institutes of Health, National Institutes of Health.
Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
Stem Cell Biology, Issue 89, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells
Institutions: Erasmus MC - University Medical Center.
Fluorescent in situ
hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist
RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.
Biochemistry, Issue 88, Fluorescent in situ hybridization (FISH), combined DNA-RNA FISH, ES cell, cytogenetics, single cell analysis, X chromosome inactivation (XCI), Xist, Bacterial artificial chromosome (BAC), DNA-probe, Rnf12
Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g.,
in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells
Institutions: University Hospital Tuebingen.
The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.
Genetics, Issue 93, mRNA synthesis, in vitro transcription, modification, transfection, protein synthesis, eGFP, flow cytometry
Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture
Institutions: University of Texas at Tyler, USDA ARS.
The glassy-winged sharpshooter (Homalodisca vitripennis
) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa),
a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) of grapevine. Pierce’s disease is economically damaging; thus, H. vitripennis
have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01
(HoCV-01) has been associated with an increased mortality in H. vitripennis
populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis
cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis
cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis
cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.
Infection, Issue 91, Homalodisca vitripennis, Homalodisca coagulata virus-01, cell culture, Pierce’s disease of grapevine, Xylella fastidiosa, Dicistroviridae
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System
Institutions: Duke-NUS Graduate Medical School, Nanyang Technological University.
Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.
Developmental Biology, Issue 96, Neuroscience, Channelrhodopsin-2, Co-culture, Neurons, Astrocytes, induced Pluripotent Stem Cells, Neural progenitors, Differentiation, Cell culture, Cortex
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis
Institutions: NYU Hospital for Joint Diseases, New York University Medical Center.
Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo
. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g.
Safranin O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.
Bioengineering, Issue 84, Mouse, Cartilage, Surgery, Osteoarthritis, degenerative arthritis, progranulin, destabilization of medial meniscus (DMM)
Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects
Institutions: Medical Center, University of California San Francisco.
The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro
and in vivo1, 2
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo
tracking with non-invasive MR imaging techniques4
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.
Cellular Biology, Issue 38, Stem cells, cartilage defect, agarose, scaffold, tissue engineering, implantation, MASI
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
Institutions: Bio-Rad Laboratories.
The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.
Cellular Biology, Issue 38, Cell Counting, Gene Silencing, siRNA, Namalwa Cells, IL4, Gene Expression, Electroporation, Real Time PCR
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Mechanical Stimulation of Chondrocyte-agarose Hydrogels
Institutions: Queen's University , Queen's University .
Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo
. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2
. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3
. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8
. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.
Cellular Biology, Issue 68, Tissue Engineering, Mechanical Stimulation, Chondrocytes, Agarose, Cartilage
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
Institutions: Klinikum rechts der Isar der Technischen Universität München, Klinikum rechts der Isar der Technischen Universität München, Klinikum rechts der Isar der Technischen Universität München, Uniklinik Köln.
Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1
. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4
. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5
. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6
. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro
. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10
. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro
and their seeding onto a 3D-matrix (Chondro-Gide
, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.
Biomedical Engineering, Issue 75, Medicine, Anatomy, Physiology, Cellular Biology, Molecular Biology, Tissue Engineering, Surgery, Autologous chondrocyte implantation, matrix-assisted, matrix, collagen scaffold, chondral lesion, cartilage, rabbit, experimental, cartilage defects, cartilage repair, regenerative therapy, chondrocytes, cell culture, isolation, transplantation, animal model
Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
Institutions: Klinikum rechts der Isar der Technischen Universität München, Klinikum rechts der Isar der Technischen Universität München, Klinikum rechts der Isar der Technischen Universität München, Uniklinik Köln.
The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1
. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2
. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8
. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10
. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11
combines bone grafting with current approaches in Tissue Engineering 5,6
. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro
and in vivo
studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14
. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18
. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21
and even first human trials 22
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro
-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.
Biomedical Engineering, Issue 75, Medicine, Anatomy, Physiology, Cellular Biology, Molecular Biology, Stem Cell Biology, Tissue Engineering, Surgery, Mesenchymal stem cells, fibrin clot, cartilage, osteochondral defect, rabbit, experimental, subchondral bone, knee injury, bone grafting, regenerative therapy, chondrocytes, cell culture, isolation, transplantation, animal model
Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering
Institutions: The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, VA Medical Center, Providence, RI, University of Texas Southwestern Medical Center .
We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.
Bioengineering, Issue 74, Biomedical Engineering, Biophysics, Cellular Biology, Medicine, Anatomy, Physiology, Cell Engineering, Bioreactors, Culture Techniques, Cell Engineering, Tissue Engineering, compression loads, shear loads, Tissues, bioreactor, mechanical loading, compression, shear, musculoskeletal, cartilage, bone, transplantation, cell culture
Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Institutions: Cytori Therapeutics Inc, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA.
In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.
Cellular Biology, Issue 79, Adipose Tissue, Stem Cells, Humans, Cell Biology, biology (general), enzymatic digestion, collagenase, cell isolation, Stromal Vascular Fraction (SVF), Adipose-derived Stem Cells, ASCs, lipoaspirate, liposuction
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Institutions: California Institute of Technology, California Institute of Technology, Massachusetts Institute of Technology, University of Minnesota.
Ideal cell-free expression systems can theoretically emulate an in vivo
cellular environment in a controlled in vitro
This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3
To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo
cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli
based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5
The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli
, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6
Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7
The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8
Accompanying mathematical models are available.9,10
The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
Cellular Biology, Issue 79, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics