Name: studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions
Uploaded: 2016-04-21T13:00:00
Description: Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

The presented work was supported in parts by funding from the Canadian Institutes of Health Research / Fragile X Research Foundation of Canada partnership program to RD, the Jerome Lejeune Foundation to RD, the Interdisziplin&#228;res Zentrum f&#252;r klinische Forschung of the University Erlangen-Nuremberg to RD and from the Deutsche Forschungsgemeinschaft to RD and RE. The authors wish to thank Ingrid Zenger for technical assistance with cell culture maintenance as well as Prof. M. Wegner for making a pCMV5-FLAG vector available. The authors further wish to particularly thank Nadja Schroeder for the helpful support at the video shooting.

1. Stock Solutions
<ol>
	<li>Resuspend the lyophilized active motif powder in sterile H2O to a final concentration of 2 &#181;g/&#181;l. Tap carefully for mixing.</li>
	<li>Prepare small aliquots (e.g., 12 &#181;l each, 2 &#181;l are required per reaction) and store them at -20 &#176;C.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 &#181;l growth medium including antibiotics. 
	NOTE: ...

<ol>
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Y., Chen, J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+RNA+interference-based+therapeutics+and+application+of+multi-target+small+interfering+RNAs.">Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs.</a> Nucleic Acid Ther. 24, 302-312 (2014).</li><li>DeVincenzo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise,+pitfalls+and+progress+of+RNA-interference-based+antiviral+therapy+for+respiratory+viruses.">The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses.</a> Antivir Ther. 17, 213-225 (2012).</li><li>Fellmann, C., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+RNA+interference+rules+for+silencing.">Stable RNA interference rules for silencing.</a> Nat Cell Biol. 16, 10-18 (2014).</li><li>Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+carrier+for+the+delivery+of+biologically+active+proteins+into+mammalian+cells.">A peptide carrier for the delivery of biologically active proteins into mammalian cells.</a> Nat Biotechnol. 19, 1173-1176 (2001).</li><li>Derlig, K., Gie&#223;l, A., Brandst&#228;tter, J. H., Enz, R., Dahlhaus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Characterisation+of+Simiate,+a+Novel+Protein+Linked+to+the+Fragile+X+Syndrome.">Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome.</a> PLoS One. 8, e83007 (2013).</li><li>Derlig, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simiate+is+an+Actin+binding+protein+involved+in+filopodia+dynamics+and+arborisation+of+neurons.">Simiate is an Actin binding protein involved in filopodia dynamics and arborisation of neurons.</a> Frontiers in Cellular Neuroscience. 8, (2014).</li><li>Loudet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-covalent+delivery+of+proteins+into+mammalian+cells.">Non-covalent delivery of proteins into mammalian cells.</a> Org Biomol Chem. 6, 4516-4522 (2008).</li><li>Nagel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+microspheres+as+protein+transduction+reagents.">Polymeric microspheres as protein transduction reagents.</a> Mol Cell Proteomics. 13, 1543-1551 (2014).</li><li>Selim, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-osteoactivin+antibody+inhibits+osteoblast+differentiation+and+function+in+vitro.">Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro.</a> Crit Rev Eukaryot Gene Expr. 13, 265-275 (2003).</li><li>Jain, A., Brady-Kalnay, S. M., Bellamkonda, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Rho+GTPase+activity+alleviates+chondroitin+sulfate+proteoglycan-dependent+inhibition+of+neurite+extension.">Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension.</a> J Neurosci Res. 77, 299-307 (2004).</li><li>Watanabe, S., Wakasugi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+function+of+human+neuroglobin+is+correlated+with+its+guanine+nucleotide+dissociation+inhibitor+activity.">Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity.</a> Biochem Biophys Res Commun. 369, 695-700 (2008).</li><li>Eguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+nuclear+localization+signal+for+nuclear+transport+of+DNA-encapsulating+particles.">Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.</a> J Control Release. 104, 507-519 (2005).</li></ol>

Here, we present a protocol to study the significance of protein expression levels in controlling cellular functions in a dose driven manner. The protocol described allows for a fine-tuned manipulation of protein expression in various mammalian cell types, including hippocampal neurons, hence facilitating detailed studies of protein function on the cellular level.
Although RNA interference represents a well known strategy to down-regulate POIs, it has its disadvantages (cp. 14). Not...

A tight control of protein expression is essential for every living organism to command its own development as well as to react to environmental signals. Hence, a multitude of mechanisms has been invented during evolution to precisely regulate the expression level of each protein encoded by the app. 20,000 genes existing in any eukaryotic cell at any given time of its life. Taking place at different stages of protein production, regulatory mechanisms range from the management of chromatin structure, transcription and RNA handling to the direction of posttranslational protein modifications, transport and degradation.
It is therefore not surprising that malfunctions in the underlying molecular machineries and altered protein expression levels have been associated with diverse diseases such as cancer or intellectual disabilities. Indeed, looking at the outstanding complexity of neuronal development and mammalian brain function, the sensitivity of these sophisticated systems to alterations in protein expression manifests itself in several well-known intellectual deficits including Alzheimer and Parkinson disease (AD and PD) as well as autism spectrum disorders (ASD) like the Fragile X Syndrome (FXS). The latter disease is characterized by an extensive misexpression of a variety of proteins, which is due to the loss of a single translation regulating protein, FMRP (Fragile X Mental Retardation Protein)1-4. Furthermore, chromosomal rearrangements affecting the Variable charge x linked protein A (VCXA), a protein, that manages mRNA stability and translation by modifying mRNA capping5, have recently been associated with intellectual deficits, while point mutations were not identified in patients with cognitive disabilities as of now6,7, suggesting that the observed mental impairments originate from altered VCXA expression and dysregulated expression of its target proteins. In line with these findings, a study investigating whether de novo copy number variations of genes are associated with ASD established that novel gene duplications and deletions are a significant risk factor for ASD8, thus supporting the idea that elevated or diminished protein expression levels may cause intellectual deficits.
Remarkably, recent research further provided evidence that the expression level of a given protein is precisely adjusted to prevent its aggregation as a consequence of high protein amounts with almost no safety margins9. It has therefore been proposed that even small increments are sufficient to induce diseases such as AD and PD9. Although the variety of molecular machineries contributing to protein expression control suggests a complex regulation scheme in the light of these findings, a study investigating the expression level of over 5,000 mammalian genes10 demonstrated that nature preferred a more parsimonious scheme: The cellular abundance of proteins was shown to be predominantly regulated at the level of translation10, thus illustrating that the management of RNA availability mainly serves to fine-tune protein expression.
Studying the dose of proteins of interest (POIs) is therefore not only important to the understanding of the endogenous functions of a protein, but also to the investigation of many diseases and the development of therapies. Thus, last decades have seen the advance of several strategies using RNA interference to manipulate POI dosage. Though RNA interference is widely used to study protein function and is even being applied in clinical trials to treat cancer or ocular diseases as well as to pursue antiviral therapies in patients11-13, some difficulties may arise which might render the strategy impossible. For example, the seed sequence, which drives the knockdown by homology is comparable short, hence promoting off-target effects. Since highly efficient sequences are rare and need to be found among thousands of options (reviewed in 14), identifying the right sequence can be time-consuming and costly, but results may still be disappointing.
An alternative strategy is to directly target the POI by antibodies. Here, we illustrate the use of the protein carrier Chariot&#160;(manufactured by Active Motif) to reduce cellular protein availability, and the employment of three-dimensional reconstructions to study protein function following knock-down.
The active motif of Chariot, itself a 2.8 kDa peptide, is used to shuttle peptides, proteins and antibodies across membranes of mammalian cells15. The peptide associates with POIs by forming non-covalently coupled macromolecular complexes utilizing hydrophobic interactions, whereupon Chariot-POI complexes are internalized into cells in an endosome-independent manner. Importantly, Chariot was indicated to neither affect the intracellular localization of shuttled proteins, nor to exert cytotoxic effects or to affect the biological activity of its cargo15.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

In the following paragraphs, exemplary results illustrating a functional knockdown of a POI (Simiate, for further details please see 16,17) using Chariot reagent and antibody interference are presented. The findings demonstrate that diminished expression of Simiate impairs transcriptional activity, and, in a dosage dependent manner, induces apoptosis, culminating in mortality rates of over 99% if higher antibody amounts (&#62; 1 &#181;g antibody) are applied (these discoveries were publis...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

The overall goal of the following experiment is to study protein functions and the role of altered protein expression in an efficient and tightly controlled manner. This is achieved by applying antibodies to reduce the amount of functional protein available to the cell. To accomplish this, antibodies are coupled to a peptide named Chariot, which mediates a passage across cellular membranes.Next, cellular structures of interest are imaged and 3D-reconstructed in order to evaluate effects of the treatment. The results reveal that Chariot peptides are particularly well-suited to target nuclear proteins by antibody interference. The main advantages of this technique over existing methods like RNA interference are that the antibodies shuttled into the cell are effective immediately, and that the researcher may strictly control the degree of interference by adjusting antibody dosages.Though this method can provide insight into protein functions in many cell types, it is particularly interesting for targeting proteins in primary neuronal cultures, which are very sensitive to treatments but tolerate Chariot very well. In preparation, resuspend lyophilized Chariot peptide powder in sterile water at two micrograms per microliter. Tap the tube carefully to mix the powder into solution.Then prepare small working aliquots, just two microliters are needed for each reaction, and store the aliquots at 20 degrees Celsius. Next, prepare mammalian cells for transfection by seeding them to a 24-well plate with half a milliliter of medium containing antibiotics. When using neurons, seed the cells only in the two central rows and at a low density.The empty wells are useful later. Be quick. Cultured neurons should not be out of the incubator for more than a few minutes.Until the neurons are at the desired developmental stage, exchange about one quarter to one half of their medium twice a week. Non-neuronal cultures should be used when the cells are at 50%confluence. The following procedure is shown for six tests and six control samples.For each cargo, prepare a dilution of 0.1 to 2 micrograms in 50 microliters of 1X PBS per sample. Macromolecular complexes of pre-bound primary and secondary antibodies can be used as cargo as well. Mix the solution with a gentle vortex, followed by a brief centrifugation.Then dilute two microliters of Chariot stock solution in 50 microliters of PBS for each reaction. Use separate tubes for every sample in order to prevent self-association of Chariot. Now, combine the prepared proteins in the Chariot dilutions.Mix and spin the combined proteins. Incubate the sample for 30 minutes at room temperature to allow for the formation of Chariot cargo complexes. The optimal protein peptide amount depends on the cargo and the cell type used.Therefore, this amount should be determined empirically. In advance, pre-warm some growth medium with no additives as well as 1X PBS containing 0.5 millimolar magnesium chloride and calcium chloride to 37 degrees Celsius. Now, to begin, dilute the prepared Chariot complexes with 100 microliters of medium.Remove the medium from the plated cells and transfer to the empty wells for reuse. Then wash the cells gently with 500 microliters of pre-warmed 1X PBS, calcium, magnesium chloride solution. Apply the medium carrying the Chariot complexes to the cells and rock the plate gently to ensure an even distribution of the solution.Quickly return the cells to the incubator and wait an hour before preceding. For neurons, after an hour of incubation, transfer the saved media to the wells. For all other cells, add serum to a final concentration of 10%Cultured neurons are very sensitive, such as to changes in media temperature and pH.Therefore, these cells should be washed and transfected gently with pre-warmed solutions and returned to the incubator as soon as possible. Continue the incubation for as long as needed, depending on the transfected cargo and the cell type. After the incubation, process the cells for subsequent analyses as usual.Fixation protocols, as well as live imaging, are both compatible with this technique. Using a laser scanning microscope with a 63X lens, take images in a distance of approximately 0.25 to 0.5 micron in order to obtain Z-stacks of sufficient detail while avoiding bleaching. The precise layer distance depends on the size of the structure to be reconstructed and needs to be determined individually.For image reconstruction, open the LSM files in Imaris. With this software, the Escape button is used to switch between Select and Navigate. Also, multiple objects can be selected by holding down the Control key.Using the Display Adjustment, go into each channel and adjust the contrast until the brightest structures reach saturation. This adjustment does not influence the surface construction but it does assist in thresholding the image. If the object to reconstructed is contained in different files, use the Edit and Add Slices function to combine the corresponding files.Next, click on the Add New Surfaces icon and select Segment only a Region of Interest. Now, adjust the margins of the selection box to fit the object of interest. Adjust the shape and tilt of the rectangular shape until all the required structures are within the box.The selection needs to be adjusted in each image all through the Z-plane. Undesired objects may be removed later on. Next, select an appropriate channel.To proceed, use the thresholding tool to reconstruct the structure of interest. The surface area detail level, or sphere diameter, should be set to match the structures being reconstructed. Depending on the signal characteristics, either absolute intensity or local contrast can be used.Generally, local contrast works better for diffuse signals. Due to the characteristics of this signal, local contrast is used to reconstruct the protein inside the nucleus. In any case, the settings needs to be adjusted separately for each signal or structure of interest.Complete the reconstruction by clicking on the green arrow at the bottom of the screen. Then adjust the object further using the Pencil tool. Carefully compare the reconstructions to the original signal, and cut and remove surfaces by tilting the images as needed so the Cut tool can accurately serve its purpose.Now, measure the volume, area, and intensities of each surface by selecting Statistics, Detailed statistics, and Average Values. If desired, color-coded statistics can be produced by selecting the Color tab of the corresponding surface and changing the selected option from Base to Statistic Coded. Employing the Take Snapshot tool, pictures can be taken for publication.If several objects within the same region are to be reconstructed, the Store Parameters function can be used to save the information. Simiate expressed in HEK293 cells was specifically detected by Simiate antibodies shuttled into the cells by Chariot peptides. Simiate antibody assemblages enter the soma as well as the nucleus, following application, where they co-localize specifically with FLAG-Simiate as marked by the yellow arrows.In a dose-dependent manner, Simiate antibodies induced massive apoptosis in HEK293 cells while control antibodies had no effect. Given that neuronal cells are very sensitive to toxic effects, the technique was applied to primary hippocampal cultures where there was no effect on the viability of neurons when comparing mock, or untreated, and Chariot-treated cells. Following the same application scheme, goat anti-guinea pig Alexa568 antibody assemblages were seen in about 83%of the cells where they occurred in somata as well as nuclei to varying extents.These observations were further supported by a 3D analysis, which was consistent with the results from the HEK293 cells. Hence, the Chariot technique can be used successfully to transfect dissociated primary hippocampal neurons. Once mastered, this technique can be done in three to six hours if performed properly.The complex formation of Chariot and the protein of interest takes about 40 minutes. To transfect the cells with these complexes, it will take 70 minutes and additional one to four hours depending on the size and properties of the cargo. Following this procedure, other methods can be performed in order to analyze the function of a protein in more detail, such as live imaging and motility assays, analysis and electrophysiological recordings.

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Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

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.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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&#160;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.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.