We sincerely thank Dr. Cindy N. Chiu for help with gel electrophoresis, and Andy Zhou and Michael Grodick for their generous donation of DNA and restriction enzyme. We gratefully acknowledge Professor J. Heath and Professor D. Prober for generous access to equipment and materials. We thank Dr. Karn Sorasaenee for helpful suggestions. We thank Mary H. Tang for creating the illustration used in the schematic overview in the video. Funding was provided by Johnson &#38; Johnson and USC Y86786.

NOTE: When working with RNA maintain a clean working environment to avoid contamination by DNase and RNase enzymes that degrade DNA and RNA. Ensure that pipette tips and tubes are DNase and RNase free. It is also helpful to wipe down lab surfaces and equipment such as pipettes, tube holders, etc. with a decontamination solution.
1. RNA Transcription with Corrole Treatment
<ol>
	<li>Prepare the corrole and inhibitor compounds in a 0.01:1 molar ratio of [complex]:[DNA]. 
	NOTE: In our case, the ratio was 4.3 fmol complex: 0.43 pmol DNA, where the DNA template used was apET-28c vector with ligA gene from E. coli under control of the T7 promoter. Other DNA with the T7 promoter are also valid candidates for running the transcription assay.
	<ol>
		<li>Dissolve Actinomycin D, triptolide, tpfc, and Ga(tpfc) in dimethyl sulfoxide (DMSO) in clean, separate containers. Obtain the final concentration of a 0.01:1 molar ratio of [complex]:[DNA] by using serial dilutions with nuclease-free water.
		<ol>
			<li>Dissolve 1 mg Actinomycin D in 1.8 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of Actinomycin D is 4.3 fmol.</li>
			<li>Dissolve 1 mg triptolide in 0.64 ml DMSO. Aliquot 1 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a second 1/1,000 dilution. 
			NOTE: The concentration of the final solution of triptolide is 4.3 fmol.</li>
			<li>Dissolve 1 mg tpfc in 2.9 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of tpfc is 4.3 fmol.</li>
			<li>Dissolve 1 mg Ga(tpfc) in 2.6 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of Ga(tpfc) is 4.3 fmol. 
			NOTE: These corroles and inhibitors are not soluble in water and must be pre-dissolved in DMSO. Small amounts of DMSO (below 1%) do not induce cytotoxicity in the cells (unpublished data).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prior to beginning the transcription reaction, prepare the necessary reagents individually or purchase them from a commercial vendor.
	<ol>
		<li>Thaw on ice all frozen reagents (refer to Table 1 for a list of frozen reagents).</li>
		<li>Allow the 10x Reaction Buffer and the 4 ribonucleotide (ATP, CTP, GTP, and UTP) solutions time to mix until they are completely dissolved in solution.</li>
		<li>Briefly centrifuge all reagents to prevent loss of material on the rim of the tube. Once thawed, store ribonucleotides on ice while keeping the 10x Reaction Buffer at RT.</li>
		<li>Combine reagents for transcription reaction at RT (refer to Table 2 for a list of reagents). 
		NOTE: The spermidine in the 10x Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice rather than at RT.</li>
		<li>Mix thoroughly by flicking the tube or pipetting the mixture up and down gently. After mixing, centrifuge briefly to collect reaction mixture at the bottom of the tube.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for a total of 2-4 hr. 
		NOTE: The necessary reaction times will vary with DNA template size and sequence. This step may require optimization for specific sequences. Shorter templates may require longer reaction times for a high yield of total RNA.</li>
		<li>Remove 4 &#181;l aliquots from each reaction after each hour and store at -20 &#176;C. Modify as needed for desired timepoints.</li>
	</ol>
	</li>
</ol>
2. RNA Spin Column
<ol>
	<li>Following the transcription reaction, purify the RNA using microcentrifuge-compatible chromatography columns. 
	NOTE: Use of RNA spin columns to purify RNA with greater than 20 bases results in greater than 80% recovery.44 
	NOTE: Column chromatography is a common and convenient method to purify RNA. Other purification methods also exist such as Lithium Chloride precipitation, phenol:chloroform extraction, isopropanol precipitation, as well as other commercially available kits.
	<ol>
		<li>Evenly resuspend the Sephadex matrix in the column buffer by vigorously inverting the column three or more times. Do this by flicking the column sharply.</li>
		<li>Remove the top cap from the column. There will be some residual Sephadex matrix in the cap; if the cap is filled with the matrix, replace the cap onto the column and repeat the previous step until the majority of the matrix is in the column.</li>
		<li>Snap off the bottom tip of the column. 
		NOTE: Some liquid may escape from the column.</li>
		<li>Pack the column.
		<ol>
			<li>Place column in a sterile (RNase and DNase free) 1.5 ml microcentrifuge tube.</li>
			<li>Centrifuge the tube in a tabletop centrifuge at 1,000 x g for 1 min.</li>
		</ol>
		</li>
		<li>Discard the 1.5 ml microcentrifuge tube with eluted buffer from the column.</li>
		<li>Immediately place the column in a new sterile 1.5 ml microcentrifuge tube and pipette the sample to the center of the column bed. Use 20-50 &#181;l of sample to avoid overloading the column.</li>
		<li>Centrifuge the tube in a tabletop centrifuge at 1,000 x g for 1 min. 
		NOTE: The eluent is the purified RNA sample.</li>
	</ol>
	</li>
</ol>
3. Agarose Gel Electrophoresis (1% Agarose Gel) 42
NOTE: Ethidium bromide fluoresces upon binding to DNA and RNA, therefore these biomolecules can be visualized with UV light by incubating them with 0.5 &#181;g/ml ethidium bromide solution.
<ol>
	<li>Prepare a 1,000x stock solution of ethidium bromide (0.5 mg/ml) by dissolving 5 mg ethidium bromide in 10 ml of water.</li>
	<li>Prepare Tris Acetate EDTA (TAE) running buffer for gel electrophoresis. To make a 50x TAE buffer combine 2.0 M Tris-acetate with 0.05 M Ethylenediaminetetraacetic acid (EDTA) and adjust the pH to 8.5.</li>
	<li>Prepare a 1% (weight/volume) agarose gel by dissolving 10 g UltraPure Agarose in 1 L 1x TAE buffer and melting the agarose with a conventional microwave oven. Once it has cooled to 50 &#176;C, add 1 ml of 1,000x ethidium bromide to the 1% agarose gel solution, mix by gently swirling or by inverting in a closed container, then pour the agarose solution into a gel-casting platform and allow the gel to solidify at RT. 
	NOTE: Ethidium bromide is a mutagen and potential carcinogen. Wear gloves and handle solutions carefully. For proper handling procedures of ethidium bromide, please see: <a href="http://www.sciencelab.com/msds.php?msdsId=9927667">http://www.sciencelab.com/msds.php?msdsId=9927667</a>.</li>
	<li>After the gel has solidified, place the set gel in the electrophoresis tank. Add enough TAE buffer to cover the gel until the wells are submerged. Check that the level of the TAE buffer is approximately 1 mm above the level of the gel. Remove the gel comb. 
	NOTE: Removing the comb after wells are submerged prevents air pockets from being trapped in the wells.</li>
	<li>Prepare the RNA samples. Combine 1 &#181;l of each purified sample aliquot with 1 &#181;l Gel Loading Buffer (or with 1 &#181;l 10x loading buffer and diluted to 10 &#181;l with nuclease-free water) and mix thoroughly by pipetting up and down with a micropipette. 
	NOTE: 10x loading buffer contains 20% Ficoll 400, 0.1 M Na2EDTA, pH 8.0, 1.0% SDS, 0.25% bromophenol blue. 0.25% xylene cyanol may also be added to the loading buffer, as it runs ~50% as fast as bromophenol blue and can be useful for monitoring very long runs.42</li>
	<li>Load the gel with each sample by carefully pipetting the solution into the bottom of each well. Take care not to leave any air bubbles or mix samples between wells. 
	NOTE: A RNA ladder can also be used to determine the size of the transcript.</li>
	<li>Attach the leads so that the DNA will migrate into the gel towards the positive lead. Set the voltage to the desired level, typically 1 to 10 V/cm of gel. Run the gel for however long is sufficient for significant separation between the RNA fragments, using the migration of dyes to monitor the progress of the separation. 
	NOTE: A 23 cm x 25 cm gel at 250 V will take approximately 1 hr, while an 8 cm x 8 cm gel at 150 V will take approximately 20 min. Smaller RNA fragments have better resolution when run at higher voltages.</li>
	<li>Turn off the power supply when the dye from the loading buffer has migrated a distance judged sufficient for separation between the RNA fragments.</li>
	<li>Image the RNA under UV light as the ethidium bromide will fluoresce.</li>
	<li>Take a picture of the image and compare fluorescence intensities of the purified RNA from each condition.</li>
</ol>
4. RNA Quantification via UV-Vis Spectroscopy
<ol>
	<li>Place 2 &#181;l H2O on a NanoDrop 2000, or similar machine, and measure the Blank.</li>
	<li>Next, place 2 &#181;l of each RNA sample, following purification, on the spectrophotometer and measure UV-Vis from a wavelength range of 200 nm to 800 nm. 
	NOTE: An A260 reading of 1.0 is equivalent to about 40 &#181;g/ml of RNA, and pure RNA has an A260/A280 ratio of 2.1.45</li>
	<li>In the case that absorbance is above 1 O.D., dilute the samples in nuclease&#8208;free water to obtain an O.D. less than 1.</li>
	<li>Use graphing software to plot the various samples and compare O.D. at 260 nm.</li>
</ol>

The authors have nothing to disclose.

This assay demonstrates that the addition of Ga(tpfc) inhibits RNA transcription comparably to the known DNA-binding anticancer complexes Actinomycin D and triptolide. The cytotoxic behavior of Ga(tpfc) (GI50 = 58.1-154.7 &#956;M) may due to its inhibitive properties. Since no transcription inhibition was observed in tpfc, the cytotoxicity of tpfc is not due to RNA transcription inhibition but is caused by other means not yet studied.
In the four transcription reactions performed, D...

Chemotherapy often involves broad-spectrum cytotoxic agents with undesired side effects and limited targeting, yet with greater understanding of cancer biology, there is an ever increasing demand for anticancer agents with higher cancer-targeting efficacy and fewer side effects.1 Human cancer cells frequently become dependent on a single activated or overexpressed oncogene for survival.2 Thus, many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Treatments that block the expression of these transforming genes are effective in eliminating cancer cells and lead to cell death.3 Transformed cells are more sensitive to disruptions in RNA transcription than are corresponding normal cells.4 Anticancer drugs that inhibit transcription are expected to selectively inhibit the expression of the oncogenes which are necessary for the cancer cell to survive.5 Consequently, RNA transcription inhibition is a useful way to identify potential anticancer drug candidates and learn more about their mechanism of action. This protocol demonstrates that Ga(tpfc) inhibits RNA transcription on the same order as the chemotherapy drugs Actinomycin D and triptolide; similar comparisons can be made using this protocol with other anticancer drug candidates. Actinomycin D is a RNA transcription inhibitor commonly used to treat gestational trophoblastic cancer, testicular cancer, Wilm&#8217;s tumor, rhabdomyosacoma, and Ewing&#8217;s sarcoma6. Actinomycin D has been used in cancer therapy for nearly fifty years since it was first approved by the FDA in 1964.Triptolide is a selective transcription inhibitor that has been investigated in vitro and in various tumor-bearing animal models for 30 years.7
The amphiphilic macrocyclic nature of corroles imparts significant advantages over other drug classes such as small molecules or biologics.8-14 The macrocyclic character allows for cellular permeability that is greater than expected for such large molecules, and they are large enough to interact with macromolecular surfaces, such as those of proteins.8 Corroles are known to form tight noncovalent complexes with biomolecules and drugs.10 In addition to the inherent cytotoxicity of the corrole framework, we have demonstrated that a sulfonated corrole acts as a carrier molecule for chemotherapeutic agents, specifically the DNA-intercalating anthracycline drug doxorubicin. When the sulfonated corrole was coadministered with doxorubicin, a 3-fold enhancement in the IC50 of doxorubicin was observed for DU-145 cells.9 The corrole framework is stable and has inherent absorbance and fluorescence properties that, when functionalized, undergo unique absorbance shifts that can be used for characterization.10 Functionalization of the scaffold does not inherently affect the photophysical properties of the corrole,9-15 but, as seen with a sulfonated corrole, selectively modifying the framework of the corrole can substantially change its biological properties.16 We previously evaluated six metallocorroles against seven human cancer cell lines. The results indicate that toxicity toward human cancer cells is dependent on the specific metal ion, as well as functional group substitution. For instance, sulfonated gallium corroles experienced high cellular uptake and penetrated selectively into the nucleus of brain metastatic prostate cancer cells (DU-145); the same corrole, though it does not penetrate into the nucleus of other cell lines, exhibits greater cytotoxicity for breast (MDA-MB-231), melanoma (SK-MEL-28), and ovarian (OVCAR-3) cancer cells than for prostate cancer.9
Initial cell-based assays indicate that these compounds show promise as anti-cancer therapeutic agents, which merits further investigation into the mechanism of action. Transcription inhibition is observed with certain organometallic complexes17-27 and we sought to examine this process as a possible mechanism for the cytotoxic behavior of the corrole family. This transcription assay provides a straightforward, inexpensive, and facile method for assessing transcription inhibition, which will lead to more detailed information about the effects of these molecules in live cells.
Here, the transcription inhibition of gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and its freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc) (Figure 1) are tested. Unlike some transition metal complexes, gallium(III) is redox inactive and therefore is not directly involved in the redox process of redox-based metabolic pathways.28 Regardless, gallium(III) does exhibit cytotoxic properties and has been investigated for therapeutic purposes. Gallium is the second most promising metal for anticancer therapeutics after platinum and has undergone many studies and investigations; nitrate and chloride gallium salts have been evaluated in clinical trials against hepatoma, lymphoma, bladder cancers, and other diseases.29-34 Gallium(III) is therefore ideal for anticancer corrole studies. Initial data show Ga(tpfc) and tpfc have low GI50, the drug concentration necessary to inhibit 50% of maximal cell proliferation, with various cancer cell lines (see Figure 2); this affirms the validity of further experiments on these two compounds to determine their inhibitive properties. We compare these compounds with the common anticancer drugs Actinomycin D and triptolide. Actinomycin D intercalates DNA, inhibits RNA elongation, and induces apoptosis in certain cell line at picomolar concentrations.6,35-37 Triptolide has shown to inhibit tumor growth; it binds to human XPB/ERCC3, a subunit of transcription factor TFIIH, leading to inhibition of RNA polymerase II activity.6-7,38-40
While it is commonly known that corroles exhibit cytotoxic properties, there exists little information about the different mechanisms arising from functionalization. Corrole inhibition of RNA transcription would offer greater insight on their interactions with biomacromolecules. Other complexes known to bind to DNA, such as dirhodium(II,II) complexes, chromium(III) complexes, ruthenium(II) polypyridyl complexes, rhodium(III) complexes, and various others, were subjected to RNA transcription assays,18-27 resulting in greater understanding of their interactions with biomacromolecules. This facile and widely available experiment is also a good initial test to assess the cytotoxicity properties of a given molecule and determine whether it merits further biological testing. The RNA transcription assay also allows for many modifications, such as varying the quantity of compound or enzymes used; varying the incubation period, reaction time and sample time points; and varying the DNA template length and sequence, among other variables of interest, thus potentially providing a large amount of data. This transcription assay is also readily available as affordable kits with all necessary reaction components provided, although components can be bought and prepared individually. In these experiments, we use a commercially available kit known to have high yield. 41
To assess transcription inhibition, we use two methods: agarose gel electrophoresis and UV-Vis spectroscopy. Agarose gel electrophoresis is a simple and effective method for separating, identifying, and purifying 0.5- to 25-kb DNA and RNA fragments.42 UV-Vis spectroscopy can be used to determine the concentration and purity of RNA.43

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Name of Material/ Equipment</td>
			<td style="width:219px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin D</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1410</td>
			<td>Store at 2-8 &#176;C , protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triptolide</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3652</td>
			<td>Store at 2-8 &#176;C , protect from light</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">nuclease-free H2O&#160;</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">AM9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">AM1334</td>
			<td>Store at &#8211;20 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Ethidium Bromide</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7637</td>
			<td>CAUTION: For proper handling procedures of ethidium bromide, please see: http://www.sciencelab.com/msds.php?msdsId=9927667</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Tris Acetate</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T6025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">UltraPure Agarose</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">16500-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mini Quick Spin RNA Columns</td>
			<td style="width:219px;">Roche Life Science</td>
			<td align="right" style="width:119px;">11814427001</td>
			<td>Store at 2-8 &#176;C , do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">1 kb DNA Ladder</td>
			<td style="width:219px;">New England Biolabs</td>
			<td style="width:119px;">N3232S</td>
			<td>Store at &#8211;20 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

RNA Transcription Qualitatively Assessed by Agarose Gel Electrophoresis
Agarose gel electrophoresis is used to image the transcribed RNA. Ethidium bromide fluoresces upon binding (&#955;em = 605 nm, &#955;ex = 210 nm, 285 nm)46 allowing imaging of RNA. Darker bands in the gel correspond to higher concentrations of RNA. If Actinomycin D, triptolide, or either corrole complex inhibits RNA transcription, the production of RNA is reduced and the ba...

Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

an-vitro-enzymatic-assay-to-measure-transcription-inhibition

Research

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Bioengineering

11.5K Görüntüleme.  California Institute of Technology.  Galyum (III) 'ün 5,10,15- (tris) pentafluorophenylcorrole ve serbest baz analog düşük mikromolar hücre sitotoksisite arzetmemiştir. Bu yazıda corroles transkripsiyon inhibisyonunu değerlendirmek için, UV-Vis spektroskopisi ile bir RNA transkripsiyon reaksiyonu, etidyum bromür ile boyanmış jel ile görüntüleme RNA ve RNA miktarının açıklar ve antikanser aday özelliklerin değerlendirilmesi için basit bir yöntem ortaya koymaktadır. 

Video: An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...