ABSTRACT
In vitro cell-based experiments are particularly important in fundamental biological research. Microscopy-based readouts to identify cellular changes in response to various stimuli are a popular choice, but gene expression analysis is essential to delineate the underlying molecular dynamics in cells. However, cell-based experiments often suffer from interexperimental variation, especially while using different readout methods. Therefore, establishment of platforms that allow for cell screening, along with parallel investigations of morphological features, as well as gene expression levels, is crucial. The droplet microarray (DMA) platform enables cell screening in hundreds of nanoliter droplets. In this study, a "Cells-to-cDNA on Chip" method is developed enabling on-chip mRNA isolation from live cells and conversion to cDNA in individual droplets of 200 nL. This novel method works efficiently to obtain cDNA from different cell numbers, down to single cell per droplet. This is the first established miniaturized on-chip strategy that enables the entire course of cell screening, phenotypic microscopy-based assessments along with mRNA isolation and its conversion to cDNA for gene expression analysis by real-time PCR on an open DMA platform. The principle demonstrated in this study sets a beginning for myriad of possible applications to obtain detailed information about the molecular dynamics in cultured cells.
Subject(s)
DNA, Complementary , Cell Line , Gene Expression , Microarray Analysis/methods , RNA, Messenger/geneticsABSTRACT
The development of novel chemically developed and physically defined surfaces and environments for cell culture and screening is important for various biological applications. The Droplet microarray (DMA) platform based on hydrophilic-superhydrophobic patterning enables high-throughput cellular screening in nanoliter volumes and on various biocompatible surfaces. Here we performed phenotypic and transcriptomic analysis of HeLa-CCL2 cells cultured on DMA, with a goal to analyze cellular response on different surfaces and culture volumes down to 3 nL, compared with conventional cell culture platforms. Our results indicate that cells cultured on four tested substrates: nanostructured nonpolymer, rough and smooth variants of poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) polymer and poly(thioether) dendrimer are compatible with cells grown in Petri dish. Cells cultured on nanostructured nonpolymer coating exhibited the closet transcriptomic resemblance to that of cells grown in Petri dish. Analysis of cells cultured in 100, 9, and 3 nL media droplets on DMA indicated that all but cells grown in 3 nL volumes had unperturbed viability with minimal alterations in the transcriptome compared with 96-well plate. Our findings demonstrate the applicability of DMA for cell-based assays and highlight the possibility of establishing regular cell culture on various biomaterial-coated substrates and in nanoliter volumes, along with routinely used cell culture platforms.