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A novel 3D cell culture method for automated drug screening

Figure 1. Global Eukaryotic Microcarrier (GEM) beads
Figure 2a. Antagonist curve, 3D freshly cultured cells, bound to GEM.
Figure 2b. Antagonist curve, 3D frozen, thawed cells, bound to GEM.
Figure 2c. Antagonist curve, 2D freshly cultured cells.
Table 1. Histamine Z’-Factor Values.
Table 2. Antagonist IC50 Values (nM).

A novel three-dimensional (3D) cell culture method, wherein cells are attached to microcarriers, is compared to traditional 2D culture methods. Results suggest that the 3D method offers similar results compared to the 2D method along with improvements in overall cost, time, in situ behaviour and automation-readiness.

by Brad Larson, Diana Berry, Brad Justice and Thomas Gainer


Over the last decade, immortalised cell lines with over-expressed drug target have been replacing purified or recombinant protein for small molecule screening applications. The reasoning behind this trend is the thought that the assays will have more biological relevance if conducted in a milieu more representative of in vivo conditions. Primary screening campaigns in particular require extensive cell culture to provide enough cells to complete the screen. Cell culturing is typically a two-dimensional (2D) method, where cells attach to a plastic surface, such as a tissue culture flask, and divide to confluence as determined by the surface area available. Proteolytic enzymes such as trypsin are then used to suspend the cells in media and the cell population is split and applied to more flasks where cells are again grown to confluence. By this iterative method, the cell population is grown to sufficient quantities necessary for a screening campaign. This process is laborious and difficult to automate, requiring significant skilled worker intervention.

A novel three-dimensional (3D) cell culture microcarrier has been identified as more convenient for the production of cells for primary and secondary cell-based screening assays. Cells are cultured on a microcarrier and suspended within the culture media, allowing for high-density cultures in reduced volumes. A variety of proteins are covalently bound to the microcarriers to promote cell adhesion and the cells can remain on the optically clear and non-autofluorescent core of the microcarriers during assays, eliminating the need for proteolytic enzymes such as trypsin.

Additionally, 3D cultured cells may be frozen in situ on the microcarrier, then thawed and used directly in downstream applications. A 3D culture method more closely mimics an in vivo environment, and studies demonstrate that the cell’s morphology, behaviour and interactions are significantly different in this realistic environment versus that of traditional flat surface cell culture methods. These differences could ultimately improve the accuracy of drug screenings, thus shortening new drug development times and reducing costs.

Here, we demonstrate use of this 3D cell culture method and compare it to traditional 2D culture methods to measure Histamine H1 G-protein-coupled receptor activity using a homogenous FRET-based assay. The entire procedure was automated in 384-well format, including cell addition, compound titration and transfer, and reagent dispensing. Validation and pharmacology data demonstrate that this 3D culture method may be used in an automated high-throughput assay system.

Microcarrier cell culture
GeneBLAzer H1-NFAT-bla HEK 293T cells (Life Technologies) contain the human Histamine Subtype 1 receptor (H1) stably integrated into the CellSensor NFAT-bla HEK 293T cell line (Life Technologies). This contains a beta-lactamase (bla) reporter gene under control of the NFAT response element. The cells were cultured on novel Global Eukaryotic Microcarrier (GEM) beads [Figure 1] from Global Cell Solutions.

The GEM are made of alginate and coated with a protein to allow cellular attachment. The unbranched polysaccharide alginate is chemically similar to components of the extracellular matrix, and provides a unique porous surface mimicking in vivo metabolic function. Sub-micron paramagnetic particles in the alginate core simplify culture manipulation. Magnets are used to easily pull the microcarriers out of suspension for sub-culturing or downstream applications.

HEK 293T cells bound to GEM were incubated in a BioLevitator benchtop incubator/bioreactor (Hamilton Company). The BioLevitator includes four independently controlled temperature- and CO2-regulated cell culture vessels, and an internal magnet controls positioning of the GEM during manual or automated procedures. Cells cultured via traditional 2D methods were also used as a control.

Comparison of cell culture methods
Cell counts were performed on freshly cultured GEM-bound, previously frozen GEM-bound and traditional 2D cultured HEK 293T cells in preparation for the GeneBLAzer Histamine H1 assay (Life Technologies). Each of the cell stocks was then diluted with assay media to 5000 cells per well. Freshly propagated cells were transferred to a new conical tube and immobilised via a magnet so that growth media could be removed and replaced with assay media. Frozen cells were immobilised via a magnet so that freezing media could be removed and replaced with assay media. Traditional 2D cultured cells were removed from the tissue culture flask using standard trypsinisation techniques and centrifuged to pellet the cells. The trypsin was subsequently removed and replaced with assay media.

All cell dispensing was performed with the MicroFlo Select Dispenser (BioTek Instruments, Inc). This dispenser uses a positive displacement peristaltic pump to dispense full incremental liquid volumes within a wide range (1μL – 10mL). Each channel is connected to an individual tip so that up to eight reagents can be simultaneously dispensed.

Stimulation or inhibition of the Histamine H1 receptor will be relayed through the NFAT pathway, causing increased or decreased production of beta-lactamase, respectively. Following addition and incubation of the beta-lactamase substrate within a well containing no bla, the substrate will remain intact. Upon excitation at 409 nm, energy is transferred from coumarin to fluorescein and emitted at 520 nm. After addition and incubation of the beta-lactamase substrate within a well containing bla, the substrate is then cleaved. Upon excitation at 409 nm, FRET does not take place, and emission is seen at 447 nm.

Agonist mode
Running the assay in agonist mode, 32μL of each cell stock were incubated in a tissue culture incubator for 20 hours at 37°C/5% CO2. Forty-eight replicates of 10μM or 0μM histamine, 8μL, were added to each of the different cell formats using the Precision Automated Microplate Pipetting System (BioTek Instruments, Inc.) The Precision combines four liquid transfer tools on one platform for dispensing into microplates, tubes and other vessels.

LiveBLAzer - FRET B/G substrate (8 μL, Life Technologies) was added to each microplate well. Following the two-hour room temperature incubation, the samples were read in fluorescence mode at 447 nm and 520 nm to detect coumarin and fluorescein signals from each assay well.  The Synergy H4 Hybrid Microplate Reader (BioTek Instruments, Inc.) combines monochromator- and filter-based fluorescence optics in one instrument for sensitive performance and flexible assay choice. Filter-based optics were used to detect coumarin and fluorescein signals, and the microplate was read from the bottom.

Antagonist 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. Thirty-two microlitres of each cell stock were incubated in a tissue culture incubator for 20 hours at 37°C/5% CO2. Antagonists were serially titrated 1:4 using the Precision Automated Microplate Pipetting System, and 4μL of a particular titrated antagonist were added to each well in the 384-well microplate. Following a 30-minute incubation (37°C/5% CO2) in the tissue culture incubator, 4μL of histamine agonist were added to each well. After a twelve-hour incubation under the same conditions, 8μL of LiveBLAzer substrate was added, and the microplate was again incubated for two hours at room temperature. Finally, the samples were read in fluorescence mode at 447 nm and 520 nm to detect coumarin and fluorescein signals from each assay well using the Synergy H4 Hybrid Microplate Reader. The instrument’s filter-based optics were used to detect coumarin and fluorescein signals, and the microplate was read from the bottom.

In Table 1, the histamine Z’-factor values, which may be calculated manually or via the Synergy H4’s Gen5 Data Analysis Software, demonstrate a statistically similar assay robustness among all culture methods. Additionally, antagonist curves [Figure 2a-c] and IC50 data [Table 2] yield pharmacologically equivalent data across all culture methods, demonstrating that each titrated antagonist covers a good range.

Summary
Cells grown on microcarrier 3D cultures provide similar results compared to traditional 2D culture methods, yet provide additional cost, time and in situ benefits. Three-dimensional methods allow for simple sub-culturing and other manipulations. Additionally, the cells bound to the microcarriers may be frozen and easily thawed for later use, and the microcarriers provide a more realistic environment than standard flat flasks. Finally, they allow for higher density cultures that use less consumables and reagents, which is highly suited for automated drug discovery applications.

Reference
aLaduron PM, Janssen PF, Gommeren W, Levsen JE: In vitro and in vivo binding characteristics of a new long-acting histamine H1 antagonist, astemizole. Mol Pharmacol 1982; 21 (2): 294-300.

The authors
Brad Larson,
BioTek Instruments, Inc., Winooski, Vermont, USA
Diana Berry and Brad Justice,
Global Cell Solutions Inc.,
Charlottesville, Virginia, USA
Thomas Gainer,
Life Technologies, Madison, Wisconsin, USA


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