Name: a novel technique for generating and observing chemiluminescence in a biological setting
Uploaded: 2017-03-09T12:30:00
Description: Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

Ethics statement: All of the in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Memorial Sloan Kettering Cancer Center (MSK) Institutional Animal Care and Use Committee (IACUC).
1. Construction of a Nebulizing Device
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	<li>Attach wood part A (12.5 x 2.5 x 1.8 cm3) upright in the center of part B (12.7 x 10.7 x 1.8 cm3) using two screws (4 x 25 mm2). Attach wood part C (11 ...

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Here, we have presented a technology that is capable of optically delineating tissue via the emission of photons created by a chemiluminescent reporter. In contrast to other, more established, technologies4,5,6,7,8,9, this chemiluminescent reporter system employs an imaging probe that is non-radioactive and facilitates detect...

In recent decades, imaging technologies have revolutionized the way that physicians diagnose and monitor disease. These imaging technologies, however, have been largely limited to whole body imaging systems, such as positron emission tomography (PET), single photon-emission computed tomography (SPECT), computed tomography (CT), and magnetic resonance imaging (MRI). Particular attention has been paid to cancer, and technological imaging breakthroughs have greatly improved the way that this disease is diagnosed and treated. Despite these advances, there is one place where these imaging technologies just don&#39;t fit: the operating room. While whole body imaging techniques can help in surgical planning, they typically lack spatial resolutions high enough to help physicians determine in real-time whether all of the tumor tissue has been removed or residual tumor tissue remains hidden at the surgical margins1. Making sure that no infiltrative tumor margins are left behind is one of the most important surgical goals, and surgeons must walk a tight-rope between rigorous and cautious tissue resection. If too much is removed, unwanted side effects for the patient are exacerbated; if too little is removed, recurrence rates are increased2,3. Therefore, it is crucial to delineate accurate tumor margins, and we believe that chemiluminescent intraoperative imaging can help to improve the accuracy of the identification of tumor margins by helping surgeons to visualize malignant tissue that could otherwise remain undetected with established techniques.
There are many imaging technologies currently being investigated for their possible utility as intraoperative imaging systems. These include &#946;- and &#947;-radiation-emitting probes4, optical fluorescence5, Raman spectroscopy6,7, and Cherenkov luminescence8,9. To date, however, none of these have become established as standard clinical tools. Optical fluorescence imaging has so far proven to be the most promising of these techniques and is therefore the most explored. While it has already been shown to be a valuable tool for many applications, it is not without its limitations. Indeed, its principal drawback is the background fluorescence generated by inherently autofluorescent biological tissue. This background autofluorescent signal is a product of the excitation of the surrounding tissue, in addition to the fluorophore, by the external light source needed for the generation of a fluorescent signal. From a practical perspective, this autofluorescence can potentially lead to low signal-to-noise ratios, which can limit the utility of this technology in the operating room.
The principal advantage of chemiluminescence imaging over fluorescence imaging is that no excitation light is necessary. As a result, there is no background autofluorescence. In chemiluminescence imaging, the excitation energy is instead generated chemically. This process produces no unintended background signal and therefore can result in higher signal-to-noise ratios. This could ultimately result in the more precise and accurate detection of surgical margins. Somewhat surprisingly, the utility of this approach as an intraoperative imaging technique has remained unexplored10. Indeed, the closest example to this technique is the oxidation of luminol by myeloperoxidase in mice11,12,13. Chemiluminescent biomedical imaging is therefore a rather unexplored area of research that could offer the following advantages: (1) minimal autofluorescence resulting in a low background signal with higher signal-to-noise ratios; (2) tunable wavelengths of chemiluminescent emissions ranging from the visible to the near-infrared; and (3) functionalizable chemiluminescent complexes that, when combined with linker technologies and targeted biomolecules that already exist, provide access to whole libraries of targeted molecular imaging probes14.
This proof-of-principle study illustrates the potential utility of chemiluminescent imaging in the biomedical setting using a ruthenium-based imaging agent. The chemiluminescent properties of this compound are well studied, with investigations dating back to the mid-1960s15. Upon chemical activation, the agent produces light at around 600 nm16, which is well suited for medical imaging purposes. The activation energy is provided by a redox reaction that leads to an excited state-which has a lifetime of 650 ns in water17-followed by the generation of photons upon relaxation of this excited state. Through the use of a specially-designed remote nebulizer, we were able to detect the compound both ex vivo and in vivo. The results of initial experiments are very promising, suggesting further investigation of this technology.

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			<td height="144" style="height:144px;width:215px;">Wood part A (12.5&#215;2.5&#215;1.8 cm)</td>
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			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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			<td style="width:119px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
			<td style="width:121px;">Comments</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">28 cm plastic cable ties</td>
			<td style="width:119px;">General Electric Inc.</td>
			<td style="width:87px;">50725</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Duct tape</td>
			<td style="width:119px;">3M Inc.</td>
			<td style="width:87px;">3939</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits w1 wire</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">w1 wire</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits p1 power</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">p1 power</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">littleBits i2 toggle switch</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">i2 toggle switch</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits 011 servo</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">011 servo</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">20 cm plastic covered wire twist ties</td>
			<td style="width:119px;">Four Star Plastics</td>
			<td style="width:87px;">71TIE8000</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="112">
			<td height="133" rowspan="2" style="height:133px;width:215px;">Tris(2,2&#8242;-bipyridyl)dichlororuthenium(II) hexahydrate</td>
			<td rowspan="2" style="width:119px;">Sigma-Aldrich Inc.</td>
			<td rowspan="2" style="width:87px;">224758</td>
			<td rowspan="2" style="width:121px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Ammonium cerium(IV) nitrate</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">22249</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Isofluorane</td>
			<td style="width:119px;">Baxter Healthcare</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">PBS</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">PBS1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Ethanol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">2854</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Triethylamine</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">T0886</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:215px;">Water</td>
			<td style="width:119px;"></td>
			<td style="width:87px;"></td>
			<td style="width:121px;">Water was purified using a Milipore Mili-Q (R &#8805; 18 M&#937;)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Female nude (outbred) mice</td>
			<td rowspan="2" style="width:119px;">Jackson Laboratories</td>
			<td rowspan="2" style="width:87px;">1929</td>
			<td rowspan="2" style="width:121px;">age 5 - 6 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Strain C57BL/6J&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">NU/J male mice at&#160;</td>
			<td style="width:119px;">Jackson Laboratories</td>
			<td style="width:87px;">2019</td>
			<td style="width:121px;">age 6 &#8211; 8 weeks</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">IVIS 200 bioluminescence reader</td>
			<td style="width:119px;">Caliper Live Science</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Live Image 4.2 software</td>
			<td style="width:119px;">Perkin-Elmer</td>
			<td style="width:87px;">128165</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Microscope slides</td>
			<td style="width:119px;">ThermoScientific</td>
			<td style="width:87px;">4951PLUS4</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

The nebulizer system described in protocol section 1 can be constructed from easily-available materials at a low cost. It is intended to be an inset for remote-triggered spraying of the reducing/oxidizing agent inside a bioluminescent reader (Figure 1). Our design allows for the safe operation of the nebulizer within the bioluminescence reader at a 14 cm distance from the lens. No fogging or blurring of the lens was observed during the operation. We selected the commercia...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

The overall goal of this protocol is to offer a new preclinical, interoperative, chemiluminescent imaging technique that can provide benefits over existing optical techniques in terms of signal-to-noise ratios and detections limits. This method can help answer key questions that have not been accessible by traditional optical imaging techniques in many fields related to the biological sciences. The main advantages of this technique are that it has a high signal-to-noise ratio and that it requires neither instant light nor radiation.So what we found is that the implications of this technique really extends towards interoperative imaging of multiple disease states including cancer because of the high signal-to-noise ratios that this technique has to offer. As incident light is not needed for this technique there are no issues with incident light absorption. Further, there is a large chemical tool box available to functionalize the chemiluminescent core molecule.To begin the procedure prepare 100 microliter standard solutions of tris(bypiridine)ruthenium(II)chloride in reverse osmosis water. Then, combine each standard with 100 microliters of a 25 millimolar solution of ammonium cerium nitrate in reverse osmosis water on a microscope slide. Open the bioluminescence reader imaging software and click initialize.In imaging mode, ensure that luminescent and photograph are checked and florescent is unchecked. Set the luminescent exposure time, binning, f/stop and emission filter parameters. Set the photograph exposure time, binning and f/stop parameters.Then set the subject height accordingly to the sample to be imaged. Set the field of view to B for a 14-centimeter distance between the camera and the sample stage. Cover the floor of the bioluminescence reader imaging chamber in the sample stage with black construction paper.Then place the first slide with the droplet of standard mixture on the sample stage with the droplet centered in the green lightbox crosshair. Next, fill the three-ounce spray bottle of a custom-built nebulizer with the 1:3 solution of triethylamine in a 50/50 solution of water and ethanol. Ensure that there are no air bubbles in the aspiration pipe.Place the nebulizer assembly in the imaging chamber with the spray nozzle pointed towards the droplet to be imaged without obstructing the camera view of the droplet. Ensure that the nebulizer power switch and power indicator are on, the magnetic toggle switch is off and the power nebulizer and remote cords are not tangled. Cover hot spots such as marks on the slide with small pieces of black construction paper.Place at least 40 centimeters of the nebulizer remote cord in the imaging chamber. Gently close the chamber door without crimping the nebulizer cords. In the instrument software, click acquire, enable autosaving and set the data file path.Initiate the imaging sequence. When the shutter opens with an audible click, switch the nebulizer toggle three times to spray three bursts of triethylamine solution onto the sample. Repeat the procedure with each standard slide to determine the minimum detectable concentration of the ruthenium imaging agent.To begin the in vivo imaging procedure, prepare 100 microliters of a solution containing between 8 and 33 nanomoles of tris(bypiridine)ruthenium(II)chloride in phosphate-buffered saline. Prepare a 25-millimolar aqueous solution of ammonium cerium nitrate. Next, intravenously inject 100 microliters of the ruthenium imaging agent into the tail vein of healthy mice.Sacrifice the mice 10 minutes after injection by carbon dioxide asphyxiation. Remove the skin from the torso with a Y cut and then remove the costal arch to expose the heart and lungs. Cut an outlet in the right atrium and inject 20 milliliters of PBS through a 24-gauge needle into the left ventricle.Cut through the belly skin to expose the kidney and liver. If the organ of interest is the liver, kidney or spleen make a visible longitudinal cut through the organ. Prepare a new imaging sequence in the bioluminescent reader software.For whole abdomen imaging, place the mouse in the imaging chamber with the open abdomen facing the camera and the head pointing towards the back of the instrument. Center the organ of interest in the green lightbox crosshair. Thoroughly wash out the spray bottle and nozzle from the nebulizer assembly and then fill the bottle with the ammonium cerium nitrate solution.Place the nebulizer in the imaging chamber with the spray nozzle directed at the imaging site. Ensure that the camera is unobstructed, the cords are not tangled and at least 40 centimeters of the remote cord are in the chamber. Close the chamber and start the imaging sequence.As the shutter opens with a click, switch the toggle three times to spray three bursts of the oxidizing agent onto the imaging site. After imaging, remove the nebulizer assembly and thoroughly rinse the spray nozzle to prevent crystallization of the ammonium cerium nitrate. For subsequent individual organ imaging and quantification, remove the mouse from the instrument and excise the inner organs starting from the open body cavity.Cut through the hind leg skin to excise muscle tissue samples. If the organ of interest is the liver, kidney or spleen cut the organ in half longitudinally. Then place the organ on a Petri dish or piece of black construction paper.Image each organ in the same way as the whole animal, rinsing the spray nozzle after each sequence. In this procedure, mice were injected with a ruthenium-based imaging agent and the tissue of interest sprayed with an oxidizer to induce chemiluminescence. The minimum detectable concentration of the imaging agent was determined to be 6.9 picomoles per centimeter squared in these conditions.Chemiluminescence in white light images of the organs of interest were obtained with a bioluminescence reader. The chemiluminescence signal was predominantly observed in the kidneys following intravenous injection indicating renal elimination of the hydrophilic tris(bypiridine)ruthenium(II)cation. The signal-to-noise ratio of the imaging agent versus PBS alone was 27:1 in the kidneys and 21:1 in the liver.Mouse popliteal lymph nodes were also evaluated with a subcutaneous injection of a more concentrated solution of the ruthenium-based imaging agent. The lymph nodes containing the imaging agent showed a significantly greater radiance than the lymph nodes containing PBS alone. Once mastered, this technique can be done in two hours if it is properly performed.While attempting this procedure, it's important to remember to exclude ambient light and to avoid contamination with ruthenium tris bpy. After its development, this technique will pave the way for researchers in the field of biomolecular imaging to explore disease states such as cancer in mice. Though this method can provide insight into questions that were not accessible by traditional optical imaging techniques, any question that could be investigated with traditional techniques potentially can be addressed with chemiluminescence.

a-novel-technique-for-generating-observing-chemiluminescence

Research

JoVE Journal

Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.