biomems and cellular biology: perspectives and applications

La capacidad de las células de cultivo ha revolucionado la comprobación de hipótesis en la célula básica y la investigación en biología molecular. Se ha convertido en una metodología estándar en la detección de drogas, la toxicología y ensayos clínicos, y es cada vez más utilizado en la medicina regenerativa. Sin embargo, la metodología de cultivo celular tradicional que consiste esencialmente en la inmersión de una gran población de células en un medio líquido homogéneo y en un sustrato plano homogéneo se ha convertido cada vez más limitantes, tanto desde una perspectiva fundamental y práctico. Tecnologías de microfabricación han permitido a los investigadores a diseñar, con el control de un micrómetro, la composición bioquímica y la topología del sustrato y la composición del medio, así como el tipo de células vecinas en el microambiente celular circundante. Además, la microtecnología es conceptualmente muy adecuado para el desarrollo de rápido, de bajo costo en sistemas in vitro que permiten a los de alto rendimiento para el cultivo y el análisis de las células en un gran número de condiciones. En esta entrevista, Albert Folch explica estas limitaciones, ¿cómo se pueden superar con la litografía blanda y la microfluídica, y se describen algunos ejemplos relevantes de la investigación en su laboratorio y las direcciones futuras.

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BioMEMS and Cellular Biology: Perspectives and Applications

BioMEMS y Biología Celular: Perspectivas y Aplicaciones

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in ...

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Video: BioMEMS and Cellular Biology: Perspectives and Applications

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Perspectives on Neuroscience

Perspectivas sobre la neurociencia

BioMEMS: Forging New Collaborations Between Biologists and Engineers

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons.  This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches.  This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments.  Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.

BioMEMS: La forja de nuevas colaboraciones entre biólogos e ingenieros

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons.  This device is compatible with ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Hacer patch pipetas y electrodos de Sharp con un extractor programable

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Recolección y criopreservación de ovocitos de hámster y de embriones de ratón

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absoluta Quantum medición del rendimiento de las muestras de polvo

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

La medición de la quimiotaxis celular con ECIS / taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Mecánica estimulación inducida por calcio Propagación de Ondas en monocapas de células: el ejemplo de las especies bovina células endoteliales corneales

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Ensayo Infinium para aplicaciones a gran escala de genotipado de SNP

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

Técnicas para inducir la senescencia celular y cuantificar

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...