Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Cell-based assays form an important part of the biological researcher’s repertoire, whether for high-throughput screening of drug candidate toxicity, querying the influence of a stimulus on motility or probing whether a treatment affects a specific metabolic pathway.
Broadly defined, a cell-based assay—in contradistinction to a biochemical assay—is one that looks at the effect of a given treatment on (typically) live cells. There are a variety of downstream readouts available to measure the effect, as well as a host of ways to structure the assay itself.
Michelle Palmer, director of discovery and preclinical research, chemical biology platform, at the Broad Institute of Harvard and MIT, talks about three nonexhaustive, overlapping “buckets” of cell-based assays. First, there are those that use artificial genetic constructs to report the result of an upstream stimulus or inhibition that induces transcription in the reporter. Second are assays that Palmer terms “biosensors,” which use nongenetic means to indicate an activity—measuring calcium flux by loading cells with a calcium-dependent dye, for example. Palmer’s third bucket is the phenotypic assay, which looks at things like cell proliferation, viability, apoptosis and senescence.
Reporter assays
In generic terms, there’s no difference between a reporter and a biosensor in the sense that you’re reporting an activity in the cell. However, reporters are typically linked to reporter-gene assays. “Reporter-gene assays are a specific kind of cell-based assay where you’ve engineered a system such that you’re incorporating the regulatory elements that control a given gene,” explains Palmer. “But you’re using an easily detectable readout like luciferase or GFP or other types of protein—enzymatic or fluorescent or chemiluminescent protein readouts.”
You may be querying the effect of an agonist or antagonist on a membrane-bound receptor or the ubiquitination of a cytosolic protein. “But ultimately your readout for that effect is that it’s turning on the pathway, ultimately up-regulating that gene expression, which is [for example] the luciferase reporter,” Palmer says.
Researchers no longer need to be crack molecular biologists to work with reporter systems specific to their gene-, protein-, pathway- or system of interest. The commercial offerings have exploded in the past five to 10 years. They run the gamut from vectors containing the upstream control elements of a gene of interest driving a luciferase reporter; to cell lines with reporter constructs already in them, awaiting introduction of a favorite receptor; to frozen, ready-to-use cell lines. There are myriad genes of interest to choose from. A host of reporters with a variety of readouts using different technologies are available. Arrays representing multiple pathways can be purchased. “To a large degree, it’s more of a reagent than it is a technique anymore,” remarked Palmer.
Special delivery
In addition to being incorporated into ready-to-use cell lines, commercial reporters are generally available as either viral vectors or DNA (plasmid) vectors (sometimes in a choice of formats), with all the accompanying advantages and disadvantages.
For example, viral transduction has a reputation for being highly reproducible and having a high rate of expression, points out Amanda Hays, research scientist at Lenexa, Kansas-based CRO XenoTech. On the other hand, when working with 20 different proteins, she finds that “it’s easier to do transient transfections on a 24-well plate rather than trying to get those 20 mutations, each one, into a viral vector.”
Retroviral vectors are subject to random generic insertion, and thus can come under the control of unknown local elements. Plasmids typically are retained as episomes. Usually, neither will be able to reproduce the endogenous regulation, “because there could be regulatory elements that are far upstream and you may be missing some tissue-specific or cell-specific regulation of a gene in the artificial system you create,” says Palmer. Newer technologies such as TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) may avoid this problem by directing the reporter to a specific location in a specific gene.
Going native
There are several label-free technologies available or pending that allow cell-based assays to be interrogated phenotypically without perturbing the system. Such functional assays avoid the need to introduce a reporter and the associated problems of transfection or transduction efficiency such as variable copy number and expression. “You measure them with a normal cell growth media and the cells are completely in their native state,” says Magnus Jansson, CSO of SymCel in Kista, Sweden. And although most reporter-based assays are one-shot or end-point assays, label-free assays are capable of giving continuous measurements of metabolic status “from hours to days or even weeks, in some instances, depending on cell type.”
SymCel’s calorimetry-based calScreener assay system monitors a culture’s heat flow down to the microwatt level. It is currently in beta testing, and Jansson expects the commercial product to ship this fall. Other label-free platforms use things like specialized optics, electrical impedance, pH or oxygen consumption to monitor changes in the culture, also in real time.
Such systems tend to engender considerable initial costs, but the outlay is often offset by a lower (in some cases nearly negligible) per-data-point cost to run the assays.
But don’t abandon reporter-based assays and throw away your multimode plate reader just yet. Label-free assays and reporter-based assays “are very much a complement to each other,” says Jansson. A reporter-gene assay measures protein expression, enabling you to quantify the response of a specific pathway. With a label-free assay “you measure the biological outcome: the metabolic rate or the metabolic response to specific stimuli. So I see them as slightly different types of assays for different purposes, really.”