This document compares the use of frozen cells versus freshly-passaged cells in label-free cell-based assays on the Corning Epic system. Three cell lines expressing different G protein-coupled receptors were tested: mu-opioid receptor, FFA1 receptor, and PKR1 receptor. The kinetic response profiles, pharmacology of reference ligands, and assay robustness were comparable between frozen and freshly-passaged cells. The results indicate that frozen cells can serve as a viable alternative to normal dividing cells for label-free cell-based assays.

1. application note
Frozen Cells Enable High Quality Label-free
Assays on the Corning® Epic® System
Author
Alice Gao and Kathy Krebs
Corning Life Sciences
Corning, New York
USA
Stéphane Parent
PerkinElmer, Inc.,
Montréal
Quebec, Canada
Introduction
Cell-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.

2. Cell lines: The three cell lines used in this study were recombinant After seeding, plates were allowed to sit in a laminar hood for 30 minutes
Chinese hamster ovary cells (CHO) from PerkinElmer’s catalog, before being placed in a humidity-controlled CO2 incubator at 37° C. After
stably expressing: overnight incubation, the media in the assay plates were replaced with
1) mu-Opioid receptor (OP3) ValiScreen® cell line assay buffer (HBSS containing calcium, magnesium, 20 mM HEPES, 0.05%
(Cat# ES-542-C) 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
2) Free Fatty Acid FFA1 receptor (GPR40) AequoScreen®
followed by the addition of test ligands. The DMR response to ligand
cell line (Cat# ES-652-A)
addition was monitored immediately for 30-40 minutes.
3) Prokineticin PKR1 receptor AequoScreen cell line
(Cat# ES-750-A) Data Analysis: The response profile/traces were obtained using Epic
Offline Viewer software. Dose responses and curve fitting were constructed
All cells were grown and maintained in F12K medium containing using GraphPad Prism® Software.
10% heat inactivated FBS, 1% Pen/Strep, and the selection agent G418
(400 µg/mL). For GPR40 and PKR1 expressing CHO cells, an additional
Results and Discussion
selective agent (250 µg/mL Zeocin) was also included in the medium.
The frozen equivalents are also available from PerkinElmer. They were: In this study, we compared three performance features between fresh and
frozen cells of the same target; 1) the kinetic response profile, 2) reference
1) cAMPZen® Frozen cells, mu-Opioid (OP3), Human Recombinant,
agonist/antagonist pharmacology and 3) assay robustness.
CHO (Cat #ES-542-CF)
2) AequoZen® Frozen cells, Free Fatty Acid FFA1 (GPR40), Human The kinetic DMR signals of the three cell lines in response to reference
Recombinant, CHO (Cat# ES-652-AF) ligands are shown in Figure 1. Overall, there were no significant changes
3) AequoZen Frozen cells, Prokineticin PKR1, Human Recombinant, in the kinetic profiles between the fresh cells and frozen cells for each of
CHO (Cat# ES-750-AF) the GPCR targets examined. For FFA1 and PKR1 expressing cell lines, the
fresh cells appear to give slightly higher response signal (about 10-15%).
These frozen cells were treated so that they are not able to propagate In contrast, the mu-Opioid expressing frozen cells performed slightly better
and were used directly in Corning® Epic® assays. under these assay conditions, yielding ~20-25% higher signal compared
to the fresh equivalent (Figure 1C). These small differences in the maximal
Corning Epic Assay Procedures: One day prior to performing the response signals are likely to be associated with the extent of cell culture
assay, fresh cells in flasks were trypsinized and harvested by centrifugation confluency in the Epic microplate. Microscopic observation confirms that
at 8000 rpm (130 g) for 3 min. The cell pellets were then re-suspended overnight culture with fresh mu-Opioid expressing cells was slightly more
in seeding medium. The resulting cell suspensions were used to seed confluent than that with frozen cells (data not shown). It is known that
Corning Epic 384-well fibronectin-coated cell-based assay microplates at some GPCRs are sensitive to contact inhibition, resulting in down regulation
8000 cells per well in a 40 μL volume. Seeding media were F12K medium of receptor activities as cell cultures reach 100% confluency. This could in
containing 10% heat inactivated FBS and 1% Pen/Strep for FFA1 and part explain the difference observed in the maximal receptor activity
PKR1 expressing cells and UltraCHO medium containing 1% Pen/Strep for between the fresh and frozen mu-Opioid expressing cells, which could
mu-Opioid expressing cells. Frozen cells were processed as follows. The be reduced or eliminated by adjusting the seeding densities for fresh or
frozen vials were briefly thawed in 37° C water bath and then transferred frozen cells.
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 Comparison of agonist and antagonist pharmacology also showed that
3 min and the cell pellets were re-suspended in the same seeding medium the frozen cells performed well in the label-free Epic® cell assays.
as their fresh equivalents. The resulting cell suspensions were used to Pharmacology data for frozen cells were not significantly different from
seed Epic microplates at 8000 cells per well in a 40 μL volume. 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.
Figure 1A Figure 1B Figure 1C
FFA1 trace 20 uM DHA PKR1 trace 1 nM OP3 trace 15 nM DAMGO
400 250 400
Fresh cells
200 Frozen cells
Response (pm)
300 300
Response (pm)
Response (pm)
150
200 200
100
100 50 100
Fresh cells Fresh cells
Frozen cells Frozen cells
0 0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 10 20 30 40
Time (min) Time (min) Time (min)
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
2

3. Figure 2A Figure 2B
Figure 2. Comparison of mu-Opioid receptor reference
OP3 cpds frozen cell OP3 cpds fresh cell ligand efficacy and potency. A: Dose response curves
400 400 obtained with frozen cells. EC50s for agonist DAMGO and
DAMGO CTOP DAMGO CTOP
Endomorphin-1 Endomorphin-1
Endomorphin-1 were 2.88 nM and 0.71 nM respectively.
IC50 for the antagonist CTOP was 25.1 nM. B: Dose
Response (pm)
300 300
Response (pm)
response curves obtained with freshly-passaged cells. EC50s
200 200 for agonist DAMGO and Endomorphin-1 were 2.94 nM and
0.88 nM respectively. IC50 for the antagonist CTOP was 36.5
100 100 nM at EC90 concentration of the agonist DAMGO. N=3
0 0
-12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5
Log [Ligand] M Log [Ligand] M
Figure 3A Figure 3B
Figure 3. Comparison of reference agonist efficacy between
FFA1 DHA PKR1, hEG-VEGF frozen and freshly-passaged cells. A: Dose response curves
500 250 obtained with FFA1 expressing cells. EC50s for agonist
Fresh cells Fresh cells Docosahexaenoid acid were 5.85 µM and 9.09 µM for frozen
200
400 and fresh cells, respectively. B: Dose response curves
Response (pm)
Response (pm)
Frozen cells Frozen cells
150 obtained with PKR1 expressing cells. EC50s for reference
300
agonist EG-VEGF were 41.4 pM and 36.5 pM for frozen
100
200 and fresh cells, respectively. N=3
50
100
0
0
-8 -7 -6 -5 -4 -3 -13 -12 -11 -10 -9 -8 -7
Log [Docosahexaenoic Acid] M Log [hEG-VEGF] M
The assessment of assay robustness with frozen cells is illustrated in Table 1. Assay robustness comparison. N=96
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 Mu-Opioid FFA1 PKR1
FFA1 expressing frozen cells, slightly higher variability in the positive con- Fresh cells
trols was evident. However, because the buffer control signals for these
frozen cells were lower than their fresh equivalents, the overall assay Z’ 0.81 0.681 0.707
robustness was equivalent between the frozen and fresh cells. Positive control 219 (±12) 358 (±14) 276 (±19)
Buffer control -4.5 (±3.1) 64 (±17) -2.7 (±8.3)
Conclusions
We have demonstrated in this study that frozen cells performed compara- Frozen cells
bly to freshly-passaged cells in the label-free Epic® cell-based assays that Z’ 0.83 0.635 0.670
evaluated G-protein coupled receptor activities. No significant differences
in the kinetic DMR profiles or reference ligand pharmacology were
Positive control 302 (±13) 353 (±26) 237 (±18)
observed between fresh cells and their frozen counterparts. The results Buffer control -4.3 (±4.0) 28 (±13) 2.9 (±7.6)
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.
Figure 4A Figure 4B Figure 4C
OP3 Robustness FFA1 robustness PKR1 Robustness
400 500 400
Fresh OP3 cells Frozen OP3 cells Fresh FFA1 cells Frozen FFA1 cells Fresh PKR1 cells Frozen PKR1 cells
400
Response (pm)
Response (pm)
300
Response (pm)
300
10 nM DAMGO
300 20 μM DHA
200 20 μM DHA 200 1 nM hEG-VEGF
10 nM DAMGO 1 nM hEG-VEGF
200
100 100
100 Buffer Controls
Buffer Controls Buffer Controls Buffer Controls
Buffer Controls Buffer controls
0 0
0
0 48 96 144 192 0 48 96 144 192 0 48 96 144 192
well number well number well number
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
3

4. PerkinElmer – Your Global Discovery Partner
PerkinElmer gives you easy access to a worldwide network of local support 3. Digan, ME, et al. (2005). Evaluation of Division-Arrested Cells for
offices, staffed by expert professionals who speak your language. You’ll Cell-Based High-Throughput Screening and Profiling. J Biomol Screen,
get expert assistance for all your questions – no matter where you are 10(6): 615-623.
located. Contact us at (800) 762-4000 or (+1) 203-925-4602, or
visit www.perkinelmer.com/ContactUs. Keeping up to date on the 4. Fang, Y, et al. (2008). Label-free cell-based assays for GPCR screening.
latest cell lines and frozen cells for label-free assays? Simply visit Combinatorial Chemistry & High Throughput Screening, 11: 357-369.
www.perkinelmer.com/assaysforlabelfree.
5. Fursov, N, et al. (2005). Improving consistency of cell-based assays
by using division-arrested cells. Assay Drug Dev. Technol. 3: 7–15.
Corning® Epic® System
Corning’s Epic system is the world’s first high-throughput, label-free 6. Kunapuli, P, et al. (2005). Application of division-arrest technology
screening system. It uses patented optical biosensor technology to to cell-based HTS: Comparison with frozen and fresh cells. Assay Drug
interrogate a broad range of biochemical and cell-based targets. Product Dev. Technol. 3: 17–26.
offerings include an Epic reader, liquid handling accessory, and 384-well
7. Masuda, Y, et al. (2002). Isolation and identification of EG-VEGF/
coated and uncoated microplates for biochemical and cell-based analysis.
prokineticins as cognate ligands for two orphan G-protein-coupled
Service offerings include assay development, screening, and hit-confirmation
receptors. Biochem. and Biophys. Res. Comm., 293(1): 396-402.
label-free services. For more information, contact us at epic@corning.com
or visit www.corning.com/epic. 8. Rickett, G, et al. (2009). Which assay technology is most appropriate
to understand the pharmacology of a ligand and how it translates
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