Sartorius eBook - 36
THE FUTURE OF DRUG DISCOVERY: LIVE 3D CELL ANALYSIS
Material and Methods
(EF1 Alpha Promoter, Puromycin selection, (fig 1)
All cell culture reagents were obtained from Life
Cat. No. 4481, prepared per Essen BioScience
Technologies unless otherwise noted. MDA-MB-
protocol). Lapatinib and Camptothecin were
231 (ATCC), SK-BR-3 (ATCC) and SKOV3 (EACC)
obtained from Sigma, and ZK164015 was
cultures were stably transfected with Incucyte®
obtained from Tocris Bioscience. Normal human
Nuclight Red Lentivirus Reagent (EF1 Alpha
dermal fibroblasts (NHDFs) were purchased from
Promoter, Puromycin selection, Cat. No. 4625,
CalTag Medsystems (Cat. No. ZHC-5102) and
prepared per Essen BioScience protocol). BT-474
MCF7 Nuclight Red cells from Essen BioScience
(ATCC) cultures were stably transfected with
(Cat. No. 4524). All cell lines were cultured in MEM
Incucyte® Cytolight Green Lentivirus Reagent
medium supplemented with 10% FBS, 1% sodium
Figure 1: Assay Workflow
TC + Stromal Cell CC
1. Coat plate
(Day 0)
Coat plate (50%
Matrigel, 40 μL/well).
Polymerize at 37° C for
30 minutes.
2a. Add cells
(Day 0)
TC + Immune Cell CC
2b. Add cells
(Day 0)
Add tumor and stromal
cells (150 μL/well, 75 μl
of each).
3. Add immune cells
(Day 3)
Add tumor cells alone
(100 μL/well). Monitor
multi-spheroid formation for 3 days.
1. 96-well micro-titer plate coated with a layer of Matrigel™
4. Add treatments
(Day 3)
Add immune cells of
interest to tumor
monoculture
(50 μL/well).
Add appropriate
treatments to
co-culture (50 μL/
well) at 4X final assay
concentration.
Monitor multi-spheroid
proliferation and death.
3. Multi-spheroid formation monitored for 3 d using
(Corning) (40 μL/well, diluted in serum-free media to a
an Incucyte® Image Analysis system (DF® Brightfield
minimum concentration of 4.5 mg/mL) and polymerized at
acquisition, 10X magnification, every 6 h)
37° C for 30 min
2. Cells of interest harvested, counted and seeded into
pre-coated 96-well plates at desired densities (150 or 100
μL/well)
a. Tumor and stromal cell co-culture: Tumor and stromal
cells seeded at 1:1 ratio (150 μL/well, 75 μL of each cell
type)
b. Tumor and immune cell co-culture: Tumor cells seeded
alone (100 μL/well)
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4. Post multi-spheroid formation, immune cells of interest
added to tumor monoculture at an optimized Effector-toTarget ratio (E:T) in a volume of 50 μL/well
5. Appropriate treatments added to co-culture assay plates
(50 μL/well) at 4X final assay concentration to achieve a
final volume of 200 μL/well
6. Multi-spheroid proliferation was monitored in Incucyte® S3
(6 h repeat scanning) for up to 10 d.
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