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

Summary

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.

Abstract

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.

Introduction

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 tox....

Protocol

1. Cell Preparation

1. Recover a vial of bioluminescent HEK293 cells from liquid nitrogen frozen stock and grow them in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 0.01 mM nonessential amino acids, 1x antibiotic-antimycotic, and 0.01 mM sodium pyruvate in a T₇₅ tissue culture flask at 37 °C and 5% CO₂.
   Note: The media and supplemental components will vary based on cell lines and therefore should be chosen accordingly.
2. Refresh medium every 2-3 days until reaching ~80% confluence.
   Note: The amount of cells required will be based on the experimental design. If....

Results

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.......

Discussion

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.

Materials

List of materials used in this article

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