IntroductionCell-based assays account for more than half of all high-throughput screens (HTS)1. These assays incorporate complex biology into the HTS process and allow the gathering of information that provides greater insight than biochemical assays into the functional behavior of drug targets. However, cells are live and dynamic entities. Instability of target protein expression, cell passage number, growth phase and differences in cell handling can often cause significant assay variability. In addition, routine maintenance and validation of cell culture stocks in preparation for screening can also be challenging even with the help of high-cost robotic systems. One alternative strategy to the use of freshly-passaged cells for cell-based assays involves the use of cryo-preserved cells. This approach separates cell preparation from drug-screening activities and can address not only the quality and variability issues with freshly-passaged cells, but also the scheduling and logistic issues facing large HTS campaigns.3,10 Historically, frozen cells have been applied successfully in many label-dependent cell-based assays, such as second messenger assays for GPCRs (i.e. calcium and cAMP), luciferase and b-lactamase reporter gene assays, as well as for high-content assays measuring intracellular trafficking events.5,6,9,10

In this report, we exploited the utility of frozen cells in a label-free cell-based assay using the Corning® Epic® technology. In cell-based assays the Corning Epic System measures the dynamic mass redistribution (DMR) that occurs within cells upon the exposure to stimuli such as drug compounds. This integrated response is pathway unbiased and enables the detection of cellular responses for endogenous as well as over-expressed targets with greater sensitivity and richer information compared to many label-dependent technologies.4 The results obtained in this study demonstrate that frozen cells are a viable alternative to freshly-passaged cells in label-free cell-based assays.

Materials and Methods

Reagents: Mu-Opioid receptor agonists DAMGO and Endomorphin-1 were purchased from Sigma-Aldrich® and Tocris Bioscience (Ellisville, Missouri, USA), respectively, and Mu-Opioid receptor antagonist CTOP was obtained from Tocris Bioscience. FFA1 (also known as GPR40) receptor agonist Docosahexanoic Acid (DHA) was purchased from Cayman Chemical® (Ann Arbor, Michigan, USA) and PKR1 receptor agonist hEG-VEGF was obtained from PeproTech® Inc. (Rocky Hill, NJ, USA). All cell culture reagents and assay buffer components were purchased from Invitrogen® (Carlsbad, California, USA), except for the UltraCHO medium which was obtained from Lonza® (Walkersville, Maryland, USA). Corning® Polypropylene plates (Cat# 3657) were used to prepare ligand solutions and Corning Epic 384-well fibronectin-coated cell-based assay microplates (Cat# 5042) were used for all assays performed in this study.

Frozen Cells Enable High Quality Label-free Assays on the Corning® Epic® System

a p p l i c a t i o n n o t e

Author

Alice Gao and Kathy KrebsCorning Life Sciences Corning, New York USA

Stéphane ParentPerkinElmer, Inc., Montréal Quebec, Canada

2

Cell lines: The three cell lines used in this study were recombinant Chinese hamster ovary cells (CHO) from PerkinElmer’s catalog, stably expressing:

1) mu-Opioid receptor (OP3) ValiScreen® cell line (Cat# ES-542-C)

2) Free Fatty Acid FFA1 receptor (GPR40) AequoScreen® cell line (Cat# ES-652-A)

3) Prokineticin PKR1 receptor AequoScreen cell line (Cat# ES-750-A)

All cells were grown and maintained in F12K medium containing 10% heat inactivated FBS, 1% Pen/Strep, and the selection agent G418 (400 µg/mL). For GPR40 and PKR1 expressing CHO cells, an additional selective agent (250 µg/mL Zeocin) was also included in the medium. The frozen equivalents are also available from PerkinElmer. They were:

1) cAMPZen® Frozen cells, mu-Opioid (OP3), Human Recombinant, CHO (Cat #ES-542-CF)

2) AequoZen® Frozen cells, Free Fatty Acid FFA1 (GPR40), Human Recombinant, CHO (Cat# ES-652-AF)

3) AequoZen Frozen cells, Prokineticin PKR1, Human Recombinant, CHO (Cat# ES-750-AF)

These frozen cells were treated so that they are not able to propagate and were used directly in Corning® Epic® assays.

Corning Epic Assay Procedures: One day prior to performing the assay, fresh cells in flasks were trypsinized and harvested by centrifugation at 8000 rpm (130 g) for 3 min. The cell pellets were then re-suspended in seeding medium. The resulting cell suspensions were used to seed Corning Epic 384-well fibronectin-coated cell-based assay microplates at 8000 cells per well in a 40 μL volume. Seeding media were F12K medium containing 10% heat inactivated FBS and 1% Pen/Strep for FFA1 and PKR1 expressing cells and UltraCHO medium containing 1% Pen/Strep for mu-Opioid expressing cells. Frozen cells were processed as follows. The frozen vials were briefly thawed in 37° C water bath and then transferred to 50-mL centrifuge tubes containing 15-20 mL seeding medium. The centrifuge tubes were then spun at centrifugation at 8000 rpm (130 g) for 3 min and the cell pellets were re-suspended in the same seeding medium as their fresh equivalents. The resulting cell suspensions were used to seed Epic microplates at 8000 cells per well in a 40 μL volume.

After seeding, plates were allowed to sit in a laminar hood for 30 minutes before being placed in a humidity-controlled CO2 incubator at 37° C. After overnight incubation, the media in the assay plates were replaced with assay buffer (HBSS containing calcium, magnesium, 20 mM HEPES, 0.05% fatty acid free BSA and 1% DMSO). The plates were allowed to equilibrate inside the Epic reader for 1 to 2.5 hours. Baseline signals were then measured followed by the addition of test ligands. The DMR response to ligand addition was monitored immediately for 30-40 minutes.

Data Analysis: The response profile/traces were obtained using Epic Offline Viewer software. Dose responses and curve fitting were constructed using GraphPad Prism® Software.

Results and Discussion

In this study, we compared three performance features between fresh and frozen cells of the same target; 1) the kinetic response profile, 2) reference agonist/antagonist pharmacology and 3) assay robustness.

The kinetic DMR signals of the three cell lines in response to reference ligands are shown in Figure 1. Overall, there were no significant changes in the kinetic profiles between the fresh cells and frozen cells for each of the GPCR targets examined. For FFA1 and PKR1 expressing cell lines, the fresh cells appear to give slightly higher response signal (about 10-15%). In contrast, the mu-Opioid expressing frozen cells performed slightly better under these assay conditions, yielding ~20-25% higher signal compared to the fresh equivalent (Figure 1C). These small differences in the maximal response signals are likely to be associated with the extent of cell culture confluency in the Epic microplate. Microscopic observation confirms that overnight culture with fresh mu-Opioid expressing cells was slightly more confluent than that with frozen cells (data not shown). It is known that some GPCRs are sensitive to contact inhibition, resulting in down regulation of receptor activities as cell cultures reach 100% confluency. This could in part explain the difference observed in the maximal receptor activity between the fresh and frozen mu-Opioid expressing cells, which could be reduced or eliminated by adjusting the seeding densities for fresh or frozen cells.

Comparison of agonist and antagonist pharmacology also showed that the frozen cells performed well in the label-free Epic® cell assays. Pharmacology data for frozen cells were not significantly different from freshly-passaged cells (Figure 2 and Figure 3). The efficacies and potencies of the reference ligands are in range with values reported in the literature2,7,8.

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Figure 1. Comparison of response profiles from cells expressing different GPCRs after stimulation with reference agonists. A) FFA1 expressing cells stimulated with 20 µM Docosahexaenoic Acid; B) Prokineticin PKR1 expressing cells stimulated with 10 nM EG-VEGF; C) Mu-Opioid expressing cells stimulated with 15 nM DAMGO. Arrows indicate the addition of the agonists. Each assay was repeated at least twice with 3 replicates for each data point. N=3

Figure 1A Figure 1B Figure 1C

3

Mu-Opioid FFA1 PKR1

Fresh cells

Z’ 0.81 0.681 0.707

Positive control 219 (±12) 358 (±14) 276 (±19)

Buffer control -4.5 (±3.1) 64 (±17) -2.7 (±8.3)

Frozen cells

Z’ 0.83 0.635 0.670

Positive control 302 (±13) 353 (±26) 237 (±18)

Buffer control -4.3 (±4.0) 28 (±13) 2.9 (±7.6)

The assessment of assay robustness with frozen cells is illustrated in Table 1 and Figure 4. As shown, all three cell assays exhibited Z’ values greater than 0.65 (Table 1), indicating that these assays are robust. For FFA1 expressing frozen cells, slightly higher variability in the positive con-trols was evident. However, because the buffer control signals for these frozen cells were lower than their fresh equivalents, the overall assay robustness was equivalent between the frozen and fresh cells.

Conclusions

We have demonstrated in this study that frozen cells performed compara-bly to freshly-passaged cells in the label-free Epic® cell-based assays that evaluated G-protein coupled receptor activities. No significant differences in the kinetic DMR profiles or reference ligand pharmacology were observed between fresh cells and their frozen counterparts. The results indicate that PerkinElmer’s frozen cells can be a viable alternative to nor-mal dividing cells as cell sources in label-free cell-based assays.

OP3 Robustness

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Figure 4 Assay performance comparison between frozen and freshly-passaged cells. A: Frozen vs. fresh mu-Opioid expressing cells. B: Frozen vs. fresh FFA1 expressing cells. C: Frozen vs. fresh Prokineticin PKR1 expressing cells. N=96

Figure 4A Figure 4B Figure 4C

Figure 2. Comparison of mu-Opioid receptor reference ligand efficacy and potency. A: Dose response curves obtained with frozen cells. EC50s for agonist DAMGO and Endomorphin-1 were 2.88 nM and 0.71 nM respectively. IC50 for the antagonist CTOP was 25.1 nM. B: Dose response curves obtained with freshly-passaged cells. EC50s for agonist DAMGO and Endomorphin-1 were 2.94 nM and 0.88 nM respectively. IC50 for the antagonist CTOP was 36.5 nM at EC90 concentration of the agonist DAMGO. N=3

Figure 3. Comparison of reference agonist efficacy between frozen and freshly-passaged cells. A: Dose response curves obtained with FFA1 expressing cells. EC50s for agonist Docosahexaenoid acid were 5.85 µM and 9.09 µM for frozen and fresh cells, respectively. B: Dose response curves obtained with PKR1 expressing cells. EC50s for reference agonist EG-VEGF were 41.4 pM and 36.5 pM for frozen and fresh cells, respectively. N=3

Figure 3A

Figure 2A

Figure 3B

Figure 2B

Table 1. Assay robustness comparison. N=96

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References

1. An, FW and Tolliday, NJ (2009) Introduction: Cell-Based Assays for High-Throughput Screening. In “Cell-Based Assays for High-Throughput Screening: Methods and Protocols” Editors: Bridget K. Wagner, Nicola J. Tolliday, Paul A. Clemons, Series: Methods in Molecular Biology, Volume 486: 1-12.

2. Briscoe, CP, et al. (2003) The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem 278: 11303-11311.

3. Digan, ME, et al. (2005). Evaluation of Division-Arrested Cells for Cell-Based High-Throughput Screening and Profiling. J Biomol Screen, 10(6): 615-623.

4. Fang, Y, et al. (2008). Label-free cell-based assays for GPCR screening. Combinatorial Chemistry & High Throughput Screening, 11: 357-369.

5. Fursov, N, et al. (2005). Improving consistency of cell-based assays by using division-arrested cells. Assay Drug Dev. Technol. 3: 7–15.

6. Kunapuli, P, et al. (2005). Application of division-arrest technology to cell-based HTS: Comparison with frozen and fresh cells. Assay Drug Dev. Technol. 3: 17–26.

7. Masuda, Y, et al. (2002). Isolation and identification of EG-VEGF/ prokineticins as cognate ligands for two orphan G-protein-coupled receptors. Biochem. and Biophys. Res. Comm., 293(1): 396-402.

8. Rickett, G, et al. (2009). Which assay technology is most appropriate to understand the pharmacology of a ligand and how it translates through to ex-vivo tissue bath studies – a case study using the mu-opioid receptor. SBS conference 2009, Poster.

9. Vasudevan, C, et al. (2005). Improving high-content-screening assay performance by using division-arrested cells. Assay Drug Dev. Technol. 3: 515–523.

10. Zaman, GJR, et al. (2007). Cryopreserved cells facilitate cell-based drug discovery. Drug Discovery Today, 12(13/14): 521-526.