®
Corning
®
CellTiter-Glo
Spheroid Microplates and Promega
3D Cell
Viability Assay provide a novel approach for high throughput screening
of multicellular spheroids.
Ana Maria P. Pardo1, Katherine Strathearn PhD1, Nicolas Andre PhD1, and Terry Riss, PhD2
1 Corning Life Sciences, Kennebunk, ME 04043, 2 Promega Corporation, Madison, WI 53711
Introduction
Consistent and reproducible results
A
B
Time Course Study
1.5×10 6
2.0×10 6
1.0×10 6
DU-145
MCF7
5.0×10 5
DU-145
1.0×
10 6
0
24
48
72
0
Time (hr)
Lum (cps)
40
200
1000
5000
10000
Lum (cps)
1000
5000
1.5×
Lum (cps)
5.0×10 4
10000
1.0×
48
24
72
Time (hr)
Time (hr)
72
Linear and sensitive assay method: Four different cell lines were plated in 96-well Spheroid Microplates at different seeding
concentrations. Spheroid growth was evaluated daily by adding CellTiter-Glo® 3D reagent straight to assay wells in a 1:1 ratio. CellTiter-Glo®
3D Cell Viability Assay demonstrated robust and consistent signals over a wide range of spheroid sizes. Representative data (n=8 wells per
cell concentration).
Uniform, single spheroid formation with multiple cell lines
•
Assay Automation
384-well Spheroid Microplate
6.0×10 5
Lum (CPS)
HT-29 (n=384)
2.0×10 5
1.0×10 5
-2
0
2
4
Summary
A
4.0×
1.5×10 5
Functionality of assay: A dose response study was used to demonstrate the functionality of the Spheroid
Microplates and CellTiter-Glo® 3D Cell Viability Assay for multicellular spheroid drug screening. Four different cell
lines were plated in 384-well Spheroid Microplates; HT-29 and A549 at 1,000 cells per well, DU-145 and MCF7
at 5,000 cells per well. Spheroids were cultured for 24 hours prior to treatment in a dose dependent manner with
Doxorubicin. Drug toxicity was evaluated over a 72 hour period by adding CellTiter-Glo® 3D reagent in a 1:1
ratio into wells. A) Dose response curves of multicellular spheroids after 72 hour treatment. B) Screen captured
shift in dose response of DU-145 spheroids over a 72 hour period. (Representative data, n=8 wells per
concentration).
Automation for spheroid screening
10 5
48 hr
Log [µM]
0
72
48
24 hr
-4
0
24
DU-145 Doxorubicin dose response
384-well Spheroid Microplate
0
Time (hr)
Scale bar 1,000 µm
4
5.0×10 4
10 6
5.0×10 5
0
B) MCF7 and A549 spheroid formation
over a 96 hour period. Spheroids
formed and cultured in 384-well
Spheroid Microplates.
2
72 hr
10 6
200
7.0×10 5
A) 72 hour spheroid cultures of HT-29
and DU-145 in 96-well Spheroid
Microplates. Cells were plated at
concentrations indicated on top.
0
2.0×10 5
2.0×10 6
0
40
0
Uniform spheroid formation in
Spheroid Microplates:
-2
2.5×10 5
HT-29 Growth Curve
High Seeding Concentration
1.0×10 5
8.0×10 5
Easy screening of multicellular spheroids: Single, uniform spheroids were generated, cultured, and assayed in Corning
Spheroid Microplates. To quantify viable cells, Promega CellTiter-Glo® 3D reagent was added directly to wells. The
microplates were read using an EnVision® Multilabel Plate Reader (PerkinElmer). The luminescent signal measured was
proportional to the number of viable cells present in the culture.
8.0×10 4
B
Consistent and reproducible results over a broad range of multicellular spheroids: Spheroid formation of four different cell lines in 96well Spheroid Microplates was evaluated by adding CellTiter-Glo® 3D reagent to wells in a 1:1 ratio. A) Spheroid formation over a 72 hour
period. All cells plated at 5,000 cells per well (data from 2 independent studies, n= 54 wells). B) Spheroid formation capabilities when using
different seeding concentrations (representative data from one study, n=8 wells per cell concentration).
9.0×10 5
48
1.2×10 5
-4
A549
24
MCF7
0
1.0×10 6
6.0×10 5
DU-145
Log [µM]
Lum (cps)
1.1×10 6
1.6×10 5
Initial seeding density per well
1.5×10 5
1.2×
A549
0
HT-29 Growth Curve
Low Seeding Concentration
Cell Proliferation study
2.0×10 5
4.0×10 4
Accuracy and linearity of data
10 6
HT-29
MCF7
5.0×10 5
Easy to use, one-step addition homogeneous assay
B
2.4×10 5
HT-29
A549
1.5×10 6
Lum (cps)
Lum (cps)
HT-29
A549
72 hr Doxorubicin dose response curve
384-well Spheroid Microplate
A
24 hr Time Point
Lum (cps)
Three dimensional (3D) cultures are recognized as more physiologically relevant
models since they provide a more accurate reflection of the intricate microenvironment
of in vivo tumor models1. However, adoption in high throughput screening (HTS)
platforms has been slow due to the limitations of the current technologies. Problems
include increased variability, low throughput, and automation issues2. The new Corning
Spheroid Microplates (Cat. Nos. 4520 and 3830) combine Corning Ultra-Low
Attachment surface and an innovative well geometry to provide an ideal tool for
generating, culturing, and assaying 3D multicellular spheroids in the same plate,
without the need for a transfer step. Another issue limiting adoption of HTS spheroid
assays is the inability of reagents to penetrate the multiple layers of 3D spheroids,
leading to inaccurate data collection3. The new Promega CellTiter-Glo® 3D Cell Viability
Assay (Cat. No. G9683) is a ready-to-use, one-step reagent specially formulated for
improved cell lysis and extraction of ATP. The assay measures ATP to quantify viable
cells, making it ideal for cell proliferation and cytotoxicity assay screening. In this study
we demonstrated the ease of generating reliable, quantifiable data for high-throughput
screening assays from multicellular spheroid tumor models using Corning Spheroid
Microplates and Promega CellTiter-Glo® 3D Cell Viability Assay.
Drug screening
Z'
%CV
0.763
7.84
HCT-116 (n=16)
0.772
7.57
A549 (n=16)
0.784
7.10
HeLa (n=16)
0.478
17.33
•
Corning Spheroid Microplates are an optimal tool for generating, culturing, and
assaying of multicellular spheroids.
•
Uniform, single spheroid formation in every well aids in maintaining
reproducibility and accuracy of assay.
•
The design of the Spheroid Microplates allows for incorporation of automation
and high-throughput screening.
Promega CellTiter-Glo® 3D Cell Viability Assay is ideal for generating quantifiable
data from multicellular spheroids.
•
Superior lytic ability and signal sensitivity allows for use over a broad range of
spheroid types and sizes.
•
Homogenous one step reagent is optimal for automation and high-throughput
screening.
0
HT-29
HCT-116
A549
HeLa
N = 384
N = 16
N = 16
N = 16
Automation friendly: To demonstrate automation friendliness, four different cell lines were plated into 384-well Spheroid Microplates using
a Multidrop™ Combi Reagent Dispenser (Thermo Scientific). After 72 hours in culture, spheroids were washed once with PBS using an
AquaMax® DW4 Microplate Washer (Molecular Devices). To quantify viable cells, a 1:1 mixture of CellTiter-Glo® 3D reagent and media were
added directly to wells. Z `values >0.5 and %CVs of less than 20%, indicate an acceptable quality assay with very little variability.
References:
1. F. Pampaloni, et al., The third dimension bridges the gap between cell culture and live tissue. Nat.
Rev. Mol. Cell Biol. 8(10), 839–845 (2007).
2. I. Vinci, et al., Advances in Establishment and Analysis of Three Dimensional Tumor Spheroid-Based
Functional Assays for Target Validation and Drug Evaluation. BMC Biology 10:29 (2012).
3. G. Mehta, et al., Opportunities and challenges for use of tumor spheroids as models to test drug
delivery and efficacy, J. Control. Release (2012), doi:10.1016/j.jconrel.2012.04.045
© 2014 Corning Incorporated