Monday, May 3, 2010

Utility of 3D cell propagation for high-throughput cell-based assays

Cell-based assays using recombinant drug targets expressed inimmortalized cell lines are today used more frequently than their biochemical counterparts in drug screening. Current cell culture methods use two-dimensional (2-D) techniques where cells attach to the microplate surface in a single monolayer. In order to maintain cell lines, complicated and time-consuming methods are used to trypsinize cells off of their growth surface, deactivate and remove the trypsin, split, and then replace cells into a new flask. This becomes increasingly difficult as scale-up is required for primary and secondary screens.

Here we show the utility of a novel three-dimensional (3-D) cell culture technique suitable for scale-up to provide primary screening quantities of cells. Cells are cultured on a magnetic alginate microcarrier, called GEM™ (Global Cell Solutions, Charlottesville, VA) coated with covalently bound coatings to promote cell adhesion, such as gelatin, laminin, or collagen. The small sub-micron magnetic particles embedded within the alginate core serve as a way to simplify culture manipulations. Since the cells grow on the microcarriers, trypsinization is not needed to split and maintain cultures. Magnetics are used to pull the microcarriers out of suspension during media changes. When cells are needed for downstream applications, an aliquot of the culture is simply aspirated from the tube used to grow the cells. As the alginate core is optically clear and non-autofluorescent, cells can remain on the microcarrier during the assay process.

In this work we show the ability of GeneBLAzer® H1-NFAT-bla HEK 293T cells, grown on the GEM™ matrix, to deliver pharmacologically relevant data using Invitrogen’s GeneBLAzer Histamine H1 Cell-Based assay to measure the activity of the Histamine H1 G-protein-coupled receptor. Cells were tested in three separate formats. Cells were cultured on the GEM™ matrix. A portion of these cells were frozen down to create a "Thaw and Use" format. The remaining cells continued to be propagated. 2D cultured cells were also tested as a control. The entire assay procedure was automated in 384-well format, including cell plating, compound titration and transfer, and reagent dispense, using simple, yet robust robotic instrumentation.



Z’-Factor and antagonist experiments were performed to validate the use of the three different cell formats with the automated assay. Z’-Factor assays were set up by adding 48 replicates of either 10 µM or 0 µM Histamine to the different cell formats, and running the assay in agonist mode.










Antagonist assays were performed with four known Histamine H1 antagonists, astemizole, pyrilamine, triprolidine, and chlorpheneramine. The H2 selective antagonist, tiotidine, was also included as an assay control. Antagonists were serially titrated 1:4 prior to addition to the 384-well assay plate.





Validation and pharmacology data demonstrate the capabilities of this novel cell culture method to be used in a high-throughput assay setting.

How do you see the use of 3D cell culturing techniques impacting high-throughput cell based assays? What can BioTek do to help you with your cell-based assays?

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