The development of robust high-throughput screening devises combining cell culture and detection systems is required for drug screening or for the applications of stem cells in therapy. In these fields, droplet-based microfluidics has recently shown avenues for controlled biological compartmentalization and high throughput cellular analysis, together with a significant reduction of the volume of reagents.
3D cultures have demonstrated improved biological functions of various mammalian cell types. In particular, the spheroids, which are tissue-like cellular aggregates, were reported to represent a physiologically relevant environment to mimic in vivo behavior. Various methods enable the formation of spheroids including hanging drops, low attachment wells etc. However, the application of these tools for high content screening is hindered by the limits on the number or the reproducibility of the spheroids. Moreover, these tools show limited compatibility with detection systems for in situ spheroid monitoring and analysis.
Here we demonstrate a microfluidic platform that integrates high-density spheroid formation, long-term culture and analysis. This platform is based on droplet techniques, with modifications that allow long term 3D culture at time scales relevant with drug or cellular microenvironment screening.
Nanoliter-sized aqueous droplets containing liquid agarose and hepactocytes were generated in fluorinated oil by flow focusing, in a microfabricated PDMS chip. The confined droplets were trapped in an array of 500 capillary anchors, through the reduction of their surface energy. After spheroid formation and hydrogel gelation, the oil phase was replaced with culture medium. The on-chip immobilized spheroids were cultivated and analyzed in situ during a 7 days culture period.
The individual spheroid formation kinetics was obtained by live imaging. Representative size and shape evolutions were determined and deviations from the mean behavior were identified. In situ live/dead, immuno-cytochemistry and BrdU staining were performed on-chip at various time points of the culture period. The analysis of over 10.000 spheroids indicated sustained viability and proliferation as well as improved expression of functional markers (i.e. intracellular albumin) compared to conventional 2D cultures. Moreover, the inter-spheroid analysis shed light on a significant correlation between albumin expression and morphometric parameters (i.e. spheroid index and size). The results were further validated by RT-PCR analysis on extracted spheroids from the chip. Furthermore, on chip intra-spheroids analysis was performed on over 100.000 cells. It was found a highly significant correlation between cellular organization (distance between cells) and the cell function.
Altogether, this study contributes to understanding the unique properties of 3D cultures by correlating micro-structural cues with the cell function. More importantly, this study demonstrates for the first time, the application of the droplet-based microfluidics into a robust and integrated long-term 3D culture and analysis platform, with potential applications in drug screening, stem cell and cancer research.