A team of Canadian researchers has developed a new microarray platform they claim can image cells in vivo every 20 minutes for as long as a week.
Led by scientists at McGill University in Montreal, the team designed the so-called "living microarrays" as a way to measure transcriptional changes in real time in single mammalian cells, and claims the approach could be used to study cell proliferation, differentiation, and apoptosis.
"Living microarrays are particularly suited for research on time-dependent processes, particularly where there is considerable cell heterogeneity, or where correlations between expression levels and cell morphology are involved," Sarav Rajan, a postdoc at the University of Toronto, told BioArray News this week. "Studies focusing on expression changes during the cell-cycle, cell differentiation, circadian rhythms and apoptosis would greatly benefit from using living microarrays."
Rajan, who co-authored a paper on the new platform in BMC Genomics last week, helped to design it while he was a PhD student at McGill. Researchers from McGill, the Genome Quebec Innovation Centre in Montreal, and the Ontario Institute for Cancer Research in Toronto contributed to its development.
According to the paper, the living microarray approach is meant to overcome drawbacks in existing technology platforms. "Endpoint measurements obtained from methods [such as DNA microarrays, RT-PCR, and other methods] that pool populations of cells are not amenable to studying time-dependent processes that show cell heterogeneity [because they] characterize a response skewed by a subset of cells in the population," they wrote.
Rajan said this week that techniques such as DNA arrays are "well-suited for measuring changes in state, such as expression levels before and after drug treatment," but that efforts to profile time-dependent behavior — for instance during muscle differentiation — were more challenging.
"We … needed a method to measure expression levels repeatedly from single living cells and relate these measurements to changes in morphology," Rajan said.
The researchers found an answer by constructing reverse-transfection cell arrays, in which transfection complexes containing reporter constructs are spotted at defined locations on a solid substrate.
According to the paper, the platform requires researchers to overlay a monolayer of adherent cells, and those that adhere to a specific spot become transfected and express a fluorescent reporter protein under the control of the promoter of interest.
They then take single-cell expression measurements by segmenting imaged spots, and use automated image analyzers to quantify the amount of intracellular fluorescence.
Rajan said reverse-transfection cell arrays have in the past enabled the transfection of specific cells with small interfering RNA and over-expression constructs. Such approaches have been "very beneficial" towards the understanding of biological systems," he said, but most are "largely centered on end-point measurements."
Instead, his team chose to extend the reverse-transfection cell-array approach by incorporating continuous expression measurements for hundreds of genes in live cells.
To accomplish this, transfection complexes were arrayed on chambered Nunc coverglass slides using a GeSim nanoplotter equipped with nano piezo-tips. Using a square pattern, the authors were able to print between 600 and 1,000 spots on the chambered coverglass slide, though they claimed the density could "easily be doubled by spotting in a checkerboard layout or using a substrate with a larger surface area."
Rajan said that any solid substrates through which cells can be imaged are suitable for hosting living microarrays. "For instance, we have also produced arrays on 10 centimeter cell culture dishes, which can easily reach densities in the thousands of constructs," he said. "The limitation then becomes the speed at which the array can be imaged, but improvements in stage control and autofocus routines have made this a tractable problem."
In the paper, the authors discussed using the living arrays to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a slide, and then imaging the clusters.
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According to the paper, the team used automated microscopy to serially acquire high-resolution images of live cells at each transfected spot. They repeated the scan continuously to generate time-lapse videos of fluorescence changes for each transfected reporter construct.
The authors claim that they were able to image each transfected cluster of cells "every 20 minutes over periods as long as 7 days," noting that this ability has enabled them to "extend population measurements by evaluating the dynamics with which a given cell will change its expression in response to a stimulus."
According to the paper, the authors used a fast-maturing, destabilized, and nuclear-localized reporter to automatically segment and measure promoter activity in the cells. The authors tested the platform with synthetic drug-inducible promoters over 24 hours that they said showed "robust induction" during the entire period.
The researchers also unintentionally found that the imaged cells displayed "substantial heterogeneity" to how they responded to the applied treatment. According to the authors, this included a "large proportion" of transfected cells that did not respond at all.
For instance, reporter studies on the GH gene promoter revealed that when activated, only 25 percent of cells displayed a sustained response, while 50 percent showed only a transient one, and 25 percent were not induced at all, the authors noted.
They say these results suggest that "within populations of cultured cells and perhaps normal tissues, there exists a mixture of cells that have different capacities to respond to external stimuli."
The findings have also yielded "questions that merit further investigation," including "whether this heterogeneity reflects the presence of distinct subpopulations of cells, or results from normal fluctuations in cell physiology."
High-throughput cell-screening arrays have been described for years. For example, in 2008 researchers at the Medical Biotechnology division of the VTT Technical Research Centre in Finland discussed the developing a miniaturized next-generation approach to microarray-based cell screening (BAN 9/16/2008).
While the authors did not comment on VTT's approach, they argued in the paper that existing methods of measuring transcription in large populations of cells suffer from "two major drawbacks."
The first is that quantifying transcription dynamics using microarrays at multiple time-points is "expensive when long processes are under study." Secondly, despite improvements in assay sensitivity, other approaches typically involve pooling mRNA from thousands of cells.
"The averaged response measured in this way is adequate for classifying different cell or tissue types, but it is not well-suited for studying processes that show cell-to-cell variation, such as cell division, differentiation, or drug responsiveness," the authors stated in the paper.
Rajan said that there are "many commercial platforms" used in high-content screening that involve multi-well formats, which are well-suited to measure the effects of various media conditions or drugs on cell function.
"Unfortunately, multiple wells also means increased variability from differing media conditions, cell seeding densities and drug concentrations," he said. "These are greatly reduced using the living microarray platform."
In its current form, the living microarray contains 600 spatially distinct clusters of cells that can be specifically transfected in a single chamber, according to Rajan. He argued that this removes many sources of well-to-well variability that are frequently associated with high-content screening, such as differences in seeding density, ligand concentration, and temperature.
In terms of future applications, the authors wrote that living microarrays could be used to screen large regions of non-coding DNA for transcriptional activity by generating a library of reporters from tiled PCR products.
"In particular, this approach could be useful for validating putative regulatory variants from genome-wide association studies," they stated.
Rajan added that the platform could "easily be converted … for siRNA screening" once candidate variants are identified by other approaches, like GWAS. He noted such a conversion could happen "either by re-spotting new constructs, or infecting the pool of reporters with a lentivirus carrying specific shRNA sequences."
Discussing commercializing the living microarray platform, Rajan said it will require more studies before the approach can be turned into a marketable product or service.
"While the technology experts we consulted felt that the technology would fill an interesting niche, further work is needed before the approach can attract commercial partners," he said.
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