Evaluation Of A Single Automated Platform For The Standardization Of Functional Cell Based Assays In Plates
The use of cell-based assays in high throughput drug screening as well as biologic development in both research and clinical trialsettings has grown due to their relevance to in vivosettings. Although there are an increasing number and diversity of functional cellular assays, the utility of these assays has not been fully recognized due to their highly variable nature. The variability arises from a variety of factors ranging from source of the cells, kind of assay, sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol and ultimately the bioinformatics.
With a view to standardizing the preparation of functional cellular assays in plates, the Biomek3000 automated workstation was evaluated. Untreated peripheral blood mononuclear cells (PBMCs), were fixed and permeabilized on the Biomek 3000 using a validated protocol for intracellular cytokine analysis. The versatility of the platform enabled the integration of the MultiScreen HTS Filter Manifold for non-centrifugal washing and recovery of cells in suspension. To optimize the wash protocol, combinations of manyfiltration plate formats from Millipore (membrane type, pore size) and mixing mechanisms to recover cells from the filter were also evaluated. The optimum conditions for the assay included the useof PVDF filter membranes with a 0.45mM pore size. The assay enabledcell recoveries of >80% without selective cell loss. The precisepipetting, timing, and washing of cells in suspension on a single platform, in the absence of centrifugation, greatly improved sample processing uniformity and reduced the variability inherent to functional assays as compared to the manually performed procedure. Additionally, the automation enabled a significant reduction of “hands-on”sample preparation and analysis time. The use of automation thus provides a greater degree of standardization in these functional assays, allowing for their use in applied research settings as surrogate markers of efficacy or activity.