Name: autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity
Uploaded: 2013-10-28T20:00:00
Description: Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

These research efforts were supported by the National Science Foundation Division of Chemical, Bioengineering, Environmental, and Transport (CBET) Systems under award numbers CBET-0853780 and CBET-1159344 and the National Institutes of Health, National Institute of Environmental Health Sciences (NIEHS) under award number 1R43ES022567-01, and the National Cancer Institute, Cancer Imaging Program under award number CA127745-01. The IVIS Lumina instrument used in this work was obtained from the U.S. Army Defense University Research Instrumentation Program.

1. Cell Preparation
<ol>
	<li>Recover a vial of bioluminescent HEK293 cells from liquid nitrogen frozen stock and grow them in Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum, 0.01 mM nonessential amino acids, 1x&#160;antibiotic-antimycotic, and 0.01 mM sodium pyruvate in a T75 tissue culture flask at 37 &#176;C and 5% CO2. 
	Note: The media and supplemental components will vary based on cell lines and therefore should be chosen accordingly.</li>
	<l...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

This method demonstrates the use of autonomously bioluminescent mammalian cells as an in vitro cytotoxicity screening assay that allows live cells to be continuously monitored over their lifetime. This protocol is flexible and can be modified to accommodate specific experimental conditions as required. For example, the experiment presented here is suitable for tracking acute toxic effects, but can be adapted to assess slow-acting or long term effects by repeatedly imaging at increased time intervals (i.e.</e...

In the U.S., pharmaceuticals and other products intended for human consumption require extensive assessment before they are approved for consumer use by the Food and Drug Administration. The financial burden for performing this testing is placed on the developer1, which substantially increases the cost of new compound development and therefore translates into an increased cost for consumers. While traditionally much of this screening has utilized animal subjects to act as proxies for human hosts, this has proven to be a large financial burden, with an estimated $2.8 billion spent annually on ADME/Tox (adsorption, distribution, metabolism, excretion, and toxicity) screening alone2&#160;and mounting evidence suggesting that animal models cannot reliably predict human toxicological responses3. Therefore, in vitro human cell culture-based testing has gained popularity over the past two decades because of its relatively lower cost, higher throughput, and better representation of human bioavailability and toxicology4. Current cell culture-based toxicity screening methods employ various endpoints, such as the measurement of ATP levels, screening the activity of endogenously available cytoplasmic enzymes, probing the integrity of the cellular membrane, or tracking the level of mitochondrial activity, to evaluate cellular viability5,6. However, regardless of the endpoint chosen, these methods all require the destruction of the sample before measurements can be taken, thus only producing data at a single time point. As a result, large numbers of samples need to be prepared and treated in parallel for basic toxicological kinetics studies, again adding to the cost and labor required for new compound development. Alternatively, assays using secreted luciferase such as Gaussia luciferase7, Vargula luciferase8, and Metridia luciferase9 have been developed that eliminate the need for cell lysis and require a fraction of the media for endpoint measurement, however, these are still limited to sampling at predetermined time points and also require the addition of exogenous light-activating substrates.
To avoid the detriment of requisite sample destruction as well as to eliminate the cost of substrates, a human cell line has been engineered that expresses the full bacterial bioluminescence (lux) gene cassette (luxCDABEfrp) to allow for continuous monitoring of live cells that is similar to fluorescent-dye based live cell imaging, but without the additional photon-activating and microscopic investigation procedures. This cell line is capable of constitutively producing an optical signal for continuous, direct detection without the need for external stimulation, thus avoiding destruction of the sample. Mechanistically, the bioluminescent signal generated from these cells results when the luxAB-formed luciferase enzyme catalyzes the oxidation of a long chain fatty aldehyde (synthesized and regenerated by the luxCDE gene products using endogenous substrates) in the presence of reduced riboflavin phosphate (FMNH2, which is recycled from FMN by the frp gene product) and molecular oxygen10. Expression of the lux cassette in the host cell therefore enables light to be produced and detected without cellular destruction or exogenous substrate addition. Similarly, the interaction between the lux genes and the endogenously available FMN, and O2 cosubstrates, and the requirement for maintenance of an environment that can support the conversion of FMN to FMNH2, ensures that the resulting bioluminescent signal can only be detected from living, metabolically active cells.
These requirements have previously been exploited to demonstrate that lux-based bioluminescent output correlates strongly with cellular population size11 and that toxic compound exposure impairs autobioluminescent production in a dose-response fashion12. Here we use a previously characterized autobioluminescent human embryonic kidney (HEK293) cell line11 to demonstrate the automated toxicological screening of an antibiotic of the bleomycin family with known DNA damaging activity as a representative example to validate the application of autobioluminescent mammalian cells for toxicity testing.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

In this study, the dynamics of autobioluminescent HEK293 cells were monitored continuously over a 24 hr period in response to antibiotic exposure (Figure 1). The toxic effects of this antibiotic, which is a member of the bleomycin family known to kill living cells by binding to and cleaving DNA13, were demonstrated via a decrease in bioluminescent production compared to untreated cells, which can be directly visualized through the pseudocolor images (Figure 2). The autobiolumi...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

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