Label-free, cell-based assays using clonetics cell systems and MDS Analytical Technologies’ CellKey System

Introduction

Label-free, cell-based assay platforms are innovative tools for assessing the integrated functional responses of cells to drug-like compounds.

The CellKey System is an impedance-based, label-free technology with proven application throughout the early drug discovery process. The system’s high sensitivity enables analysis of endogenous receptors in physiologically relevant and clinically predictive cell systems. Clonetics Primary Cells and Media provide a validated and reliable source of physiologically relevant cells for drug discovery applications, such as target identification and receptor panning.

The CellKey System, available in 96-well and 384-well formats, measures changes in the impedance (dZ) of a cell layer that occur in response to receptor stimulation. Cells are seeded onto a custom CellKey microplate that contains electrodes at the bottom of the wells. The instrument applies small voltages at low frequencies, and these voltages induce extracellular currents (iec) that pass around individual cells in the monolayer. At high frequencies, they induce transcellular currents (itc) that penetrate the cellular membrane.

Changes in impedance due to extracellular currents (dZiec) and transcellular currents (dZitc) are reported kinetically for each well (Figure 1A). When cells are exposed to a stimulus, such as a receptor ligand, signal transduction events occur that lead to a variety of cellular responses, including changes in cell adherence, cell shape and volume, and cell-cell interaction. Each of these physiological responses can be linked to receptor stimulation through classical signalling pathways that result, for example, in changes to cytoskeletal organization.

Figure 1: A: The CellKey System applies constant voltage, producing current that flows around and between cells (extracellular current, iec) and through cells (transcellular current, itc). Impedance (Z) is the ratio of voltage (V) to current (I), as described by Ohm’s Law. B: Kinetic response profile depicting changes in impedance related to extracellular current (dZiec) and changes in impedance related to transcellular current (dZitc) measurements, over time.

These cellular changes, individually or collectively, affect the flow of extracellular and transcellular current, and therefore, affect the magnitude and characteristics of the measured impedance (Figure 1B).

Materials and methods

CellKey System target identification - Clonetics HSMMHuman Skeletal Muscle Myoblasts

Clonetics HSMM-Human Skeletal Muscle Myoblasts were seeded onto CellKey Standard 96-Well microplates at a density of 40,000 cells/well and grown overnight in Clonetics SkGM-2 Skeletal Muscle Cell Growth Medium-2 under standard tissue culture conditions. SkGM-2 Medium was changed to Hanks HEPES buffer with 0.1% (w/v) BSA (HHB) 30 minutes before the start of each experiment, and cells were incubated at 37°C to allow for equilibration to the new buffer environment. Equilibrated cell plates were placed onto the system and pre-addition measurements were made for 5 minutes to obtain a baseline reading. Using onboard fluidics, receptor ligands were applied to all wells simultaneously while the instrument actively measured the impedance in each well. In these experiments, a panel of 72 commercially available ligands were tested. Measurement continued for 10 minutes after ligand addition to monitor cellular responses.

Figure 2: Assay steps. Primary cells seeded onto custom 96-well microplates were equilibrated at 37°C for 30 minutes in Hanks HEPES buffer with 0.1% BSA (HHB). Equilibrated cell plates were placed onto the system and pre-addition measurements were made for 5 minutes to obtain a baseline reading. Ligands were added to all wells simultaneously while the instrument actively measured the impedance in each well. Measurement continued for 10 minutes after ligand addition to monitor cellular responses.

CellKey System target validation in Clonetics HSMM Cells

For antagonist experiments, Clonetics HSMM Cells were pretreated with compound for 30 minutes prior to assay start. For pathway validation studies, cells were treated with 100 ng/ml pertussis toxin (PTX) (a well-known blocker of G_&$945;i-coupled G-protein coupled receptor (GPCR) signalling) overnight. All experiments were performed at 37°C.

Donor-to-donor variability - Clonetics HUVECS-Human Umbilical Vein Endothelial Cells

Clonetics HUVECS-Human Umbilical Vein Endothelial Cells from three separate donors were seeded onto CellKey Standard 96-Well microplates at a density of 35,000 cells/well and grown overnight in EGM-2 Endothelial Cell Growth Medium-2 under standard tissue culture conditions. EGM-2 Medium was changed to Hanks HEPES buffer with 0.1% (w/v) BSA (HHB) 30 minutes before the start of each experiment, and cells were incubated at 37°C to allow for equilibration to the new buffer environment. Equilibrated cell plates were placed onto the system and pre-addition measurements were made for 5 minutes to obtain a baseline reading. The cellular responses to a panel of commercially available ligands were assessed as detailed below.

Results

CellKey System target identification - Clonetics HSMM Cells

Clonetics HSMM Cells are an effective cellular model for a variety of important diseases including diabetes and muscular dystrophy. In these experiments, active endogenous receptors were identified via receptor panning, where a panel of 72 commercially available known receptor ligands were applied to the cells (a subset is shown in Figure 3). A threshold of ± 3 x SD of response to buffer (negative control) was set to distinguish significant responses (Actives) from background.

Borderline active responses were identified subjectively. As can be seen in Figure 3, Clonetics HSMM-Human Skeletal Muscle Myoblasts express a range of interesting GPCR’s and protein tyrosine kinase receptors (PTKRs).

Figure 3: CellKey System target identification - Clonetics HSMM-Human Skeletal Muscle Myoblasts. Clonetics HSMM were plated onto CellKey Standard 96-Well microplates at a density of 40,000 cells/well. A threshold of ± 3 x SD of buffer was set to distinguish significant responses from background. All experiments were performed as detailed in the Materials and Methods and conducted at 37°C. Data are n = 3 ± SD.

CellKey System target validation in Clonetics HSMM Cells

The cellular response induced by administration of the adenosine receptor agonist NECA was further validated via concentration response curves for agonists and antagonists (Figures 4A and 4B). Pertussis toxin experiments demonstrating the G_&$945;i-dependent coupling of the adenosine receptor response are shown in Figure 4C. Based on this result and the receptor pharmacology, an A1 adenosine receptor subtype is expressed in these primary cells. Interestingly, addition of the compound XAC generated cellular responses that would indicate an inverse agonist at the adenosine receptor in Clonetics HSMM Cells (Figure 4D). If confirmed, this is a rare demonstration of inverse agonism in the endogenous setting, and illustrates the power and importance of matching primary cells with label-free impedance assays.

Figure 4: CellKey System target validation in Clonetics HSMM Cells. A: and B: Validation of the cellular response induced by the adenosine receptor agonist, NECA, via concentration response curves for agonists and antagonists C: Bar graph of pertussis toxin experiments demonstrating the Gαi-dependent coupling of the adenosine receptor response. D: The compound, XAC, was observed to behave as an inverse agonist at the adenosine receptor in Clonetics HSMM. Data are n = 4 to 6, ± SD.

Active endogenous receptors analysed across three different Clonetics HUVEC-Human Umbilical Vein Endothelial Cell donors

HUVECS have become one of the most widely used primary cell types in drug discovery, due to their relative abundance and ease of isolation. HUVECs are important models for cardiovascular pharmaceutical development and vascular pathology, as well as for oncology indications related to angiogenesis. In the receptor panning experiments shown in Figure 5, Clonetics HUVECs from 3 different donors were assessed for similarities and differences in their response levels. Although donor-to-donor differences are small, clear differences can be seen. Label-free receptor panning applied to primary cells from varied sources is useful, not only in qualifying clinically viable receptor targets, but also in assigning physiologically relevant hit/miss cut-off values.

Pharmacological analysis of receptor agonists

In follow-up to the receptor panning experiments shown in Figure 5, pharmacological analyses of the endogenous Edg receptors expressed in three different Clonetics HUVEC donors (Figure 6A) and adrenergic (Isoproterenol) and purinergic (uridine 5’-triphosphate; UTP) receptors in two donors (Figure 6B) were performed. As can be seen from this data, the curves produced show good correlation between donors and produce similar EC₅₀ values. These types of studies are useful for determining the degree to which person-to-person variability may impact clinical drug efficacy.

Figure 5: Active endogenous receptors analyzed across Clonetics HUVEC-Human Umbilical Vein Endothelial Cells from three different donors. Clonetics HUVECs were plated onto CellKey Standard 96-Well microplates at a density of 35,000 cells/ well. A threshold of ± 3 x SD of buffer was set to distinguish significant responses from background. All experiments were performed as detailed in the Materials and Methods and conducted at 37°C.

Figure 6: Pharmacological analysis of receptor agonists. A: Following on from receptor panning experiments, pharmacological analyses were performed of the endogenous Edg receptors expressed in three different HUVEC donors and B: adrenergic and purinergic receptors in two donors. Data are n =4 ± SD.

Conclusions

Collaborative studies performed between Lonza and MDS Analytical Technologies matched a number of Lonza Clonetics Primary Cells with the CellKey System. Utilizing the simplified label-free workflow and the optimized cell culture protocols provided by Lonza, routine culture and analysis of important primary cell types was made simple on the CellKey System. This combination of technologies clearly demonstrates the ease of employing Clonetics Primary Cells in the discovery process in order to prioritize lead compounds earlier and more effectively. The resulting data is robust, reproducible, biologically relevant, and powerful, taking full advantage of endogenous levels of receptor targets attached to their native response pathways in cell types more closely aligned to normal tissues.

Matching the CellKey System with Lonza’s optimized Clonetics Primary Cells, Media and protocols enables:

• Simple adaptation of Clonetics Primary Cells to label-free assays
• Maximal biological relevance through analysis of native receptor pathway in primary cells
• Robust and more clinically predictive target identification and validation
• Robust and more clinically predictive pharmacology

Citations

Technical Poster: Society for Biomolecular Sciences 2009 Meeting, Lille, France.

R McGuinness, Oral Presentation: World Pharmaceutical Congress, 2009 Philadelphia, USA.

By Debra Gallant¹, Ryan McGuinness¹, Martin Brown², Kevin Mobbs²;

1 MDS Analytical Technologies (US) Sunnyvale, CA, USA; 2 Lonza, Walkersville, MD, USA