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Find video protocols related to scientific articles indexed in Pubmed.
Human-on-chip for therapy development and fundamental science.
Curr. Opin. Biotechnol.
PUBLISHED: 02-04-2014
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Organ-on-chip systems integrate microfluidic technology and living cells to study human physiology and pathophysiology. These human in vitro models are promising substitutes for animal testing, and their small scale enables precise control of culture conditions and high-throughput experiments, which would not be economically sustainable on a macroscopic level. Multiple sources of biological material are used in the development of organ-on-chips, from biopsies to stem cells. Each source has its own peculiarities and technical requirements for integration into microfluidic chips, and is suitable for specific applications. While a biopsy is the tissue of choice for the biomimetic response to ageing, induced pluripotent stem cells hold great promise for the study of genetic-related disease pathogenesis, and primary cultures can fill the gap.
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Determination of glucose flux in live myoblasts by microfluidic nanosensing and mathematical modeling.
Integr Biol (Camb)
PUBLISHED: 01-15-2014
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Glucose is the main energy source for cells in an organism and its blood concentration is tightly regulated in healthy individuals. However, impaired blood glucose control has been found in diseases such as metabolic syndrome and diabetes, and anomalous glucose utilization in cancer tissues. Dissecting the dynamics of the different phenomena involved in glucose handling (extracellular mass transport, membrane diffusion, and intracellular phosphorylation) is very relevant to identify which mechanisms are disrupted under disease conditions. In this work, we developed an effective methodology for quantitatively analyzing these phenomena in living cells. A measurement of steady-state glucose uptake is, by itself, insufficient to determine the dynamics of intracellular glucose. For this purpose, we integrated two types of measurements: cytosolic glucose concentration at the single-cell level, obtained using a cytosolic FRET nanosensor, and cell population glucose uptake, obtained without perturbing culture conditions using a microfluidic perfusion system. Microfluidics enabled accurate temporal stimulation of cells through cyclic pulses of glucose concentration at defined flow rates. We found that both, glucose uptake and phosphorylation, are linearly dependent on glucose concentration in the physiological range. Mathematical modeling enabled precise determination of the kinetic constants of membrane transport (0.27 s(-1)) and intracellular phosphorylation (2.01 s(-1)).
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Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers.
Biol. Cell
PUBLISHED: 05-23-2013
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In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel.
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Stochastic model-assisted development of efficient low-dose viral transduction in microfluidics.
Biophys. J.
PUBLISHED: 02-28-2013
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Adenoviruses are commonly used in vitro as gene transfer vectors in multiple applications. Nevertheless, issues such as low infection efficiency and toxicity effects on host cells have not been resolved yet. This work aims at developing a new versatile tool to enhance the expression of transduced genes while working at low viral doses in a sequential manner. We developed a microfluidic platform with automatically controlled sequential perfusion stages, which includes 10 independent channels. In addition, we built a stochastic mathematical model, accounting for the discrete nature of cells and viruses, to predict not only the percentage of infected cells, but also the associated infecting-virus distribution in the cell population. Microfluidic system and mathematical model were coupled to define an efficient experimental strategy. We used human foreskin fibroblasts, infected by replication-incompetent adenoviruses carrying EGFP gene, as the testing system. Cell characterization was performed through fluorescence microscopy, followed by image analysis. We explored the effect of different aspects: perfusion, multiplicity of infection, and temporal patterns of infection. We demonstrated feasibility of performing efficient viral transduction at low doses, by repeated pulses of cell-virus contact. This procedure also enhanced the exogenous gene expression in the sequential microfluidic infection system compared to a single infection at a higher, nontoxic, viral dose.
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Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions.
Biotechnol. Prog.
PUBLISHED: 02-15-2011
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Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O2 concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O2 solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O2 concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34+ cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O2 for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34+ cells at 5% O2, using 21%-O2 cultures as a control. In hypoxia we found that CD34+ cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level.
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Confidence from uncertainty--a multi-target drug screening method from robust control theory.
BMC Syst Biol
PUBLISHED: 05-26-2010
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Robustness is a recognized feature of biological systems that evolved as a defence to environmental variability. Complex diseases such as diabetes, cancer, bacterial and viral infections, exploit the same mechanisms that allow for robust behaviour in healthy conditions to ensure their own continuance. Single drug therapies, while generally potent regulators of their specific protein/gene targets, often fail to counter the robustness of the disease in question. Multi-drug therapies offer a powerful means to restore disrupted biological networks, by targeting the subsystem of interest while preventing the diseased network from reconciling through available, redundant mechanisms. Modelling techniques are needed to manage the high number of combinatorial possibilities arising in multi-drug therapeutic design, and identify synergistic targets that are robust to system uncertainty.
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Microliter-bioreactor array with buoyancy-driven stirring for human hematopoietic stem cell culture.
Biomicrofluidics
PUBLISHED: 03-15-2010
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This work presents the development of an array of bioreactors where finely controlled stirring is provided at the microliter scale (100-300 mul). The microliter-bioreactor array is useful for performing protocol optimization in up to 96 parallel experiments of hematopoietic stem cell (HSC) cultures. Exploring a wide range of experimental conditions at the microliter scale minimizes cost and labor. Once the cell culture protocol is optimized, it can be applied to large-scale bioreactors for stem cell production at the clinical level. The controlled stirring inside the wells of a standard 96-well plate is provided by buoyancy-driven thermoconvection. The temperature and velocity fields within the culture volume are determined with numerical simulations. The numerical results are verified with experimental velocity measurements using microparticle image velocimetry (muPIV) and are used to define feasible experimental conditions for stem cell cultures. To test the bioreactor arrays functionality, human umbilical cord blood-derived CD34(+) cells were cultured for 7 days at five different stirring conditions (0.24-0.58 mums) in six repeated experiments. Cells were characterized in terms of proliferation, and flow cytometry measurements of viability and CD34 expression. The microliter-bioreactor array demonstrates its ability to support HSC cultures under stirred conditions without adversely affecting the cell behavior. Because of the highly controlled operative conditions, it can be used to explore culture conditions where the mass transport of endogenous and exogenous growth factors is selectively enhanced, and cell suspension provided. While the bioreactor array was developed for culturing HSCs, its application can be extended to other cell types.
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Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.
Biotechnol. Prog.
PUBLISHED: 02-19-2009
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It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.
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Computational modeling of glucose transport in pancreatic ?-cells identifies metabolic thresholds and therapeutic targets in diabetes.
PLoS ONE
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Pancreatic ?-cell dysfunction is a diagnostic criterion of Type 2 diabetes and includes defects in glucose transport and insulin secretion. In healthy individuals, ?-cells maintain plasma glucose concentrations within a narrow range in concert with insulin action among multiple tissues. Postprandial elevations in blood glucose facilitate glucose uptake into ?-cells by diffusion through glucose transporters residing at the plasma membrane. Glucose transport is essential for glycolysis and glucose-stimulated insulin secretion. In human Type 2 diabetes and in the mouse model of obesity-associated diabetes, a marked deficiency of ?-cell glucose transporters and glucose uptake occurs with the loss of glucose-stimulated insulin secretion. Recent studies have shown that the preservation of glucose transport in ?-cells maintains normal insulin secretion and blocks the development of obesity-associated diabetes. To further elucidate the underlying mechanisms, we have constructed a computational model of human ?-cell glucose transport in health and in Type 2 diabetes, and present a systems analysis based on experimental results from human and animal studies. Our findings identify a metabolic threshold or "tipping point" whereby diminished glucose transport across the plasma membrane of ?-cells limits intracellular glucose-6-phosphate production by glucokinase. This metabolic threshold is crossed in Type 2 diabetes and results in ?-cell dysfunction including the loss of glucose stimulated insulin secretion. Our model further discriminates among molecular control points in this pathway wherein maximal therapeutic intervention is achieved.
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Flow biosensing and sampling in indirect electrochemical detection.
Biomicrofluidics
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Miniaturization in biological analyses has several advantages, such as sample volume reduction and fast response time. The integration of miniaturized biosensors within lab-on-a-chip setups under flow conditions is highly desirable, not only because it simplifies process handling but also because measurements become more robust and operator-independent. In this work, we study the integration of flow amperometric biosensors within a microfluidic platform when analyte concentration is indirectly measured. As a case study, we used a platinum miniaturized glucose biosensor, where glucose is enzymatically converted to [Formula: see text] that is oxidized at the electrode. The experimental results produced are strongly coupled to a theoretical analysis of fluid dynamic conditions affecting the electrochemical response of the sensor. We verified that the choice of the inlet flow rate is a critical parameter in flow biosensors, because it affects both glucose and [Formula: see text] transport, to and from the electrode. We identify optimal flow rate conditions for accurate sensing at high time resolution. A dimensionless theoretical analysis allows the extension of the results to other sensing systems according to fluid dynamic similarity principles. Furthermore, we developed a microfluidic design that connects a sampling unit to the biosensor, in order to decouple the sampling flow rate from that of the actual measurement.
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Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.
Langmuir
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Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 ?m width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 ?m wide lanes compared to (39 ± 7)% in 500 ?m lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 ?m, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.