Sartorius eBook - 37

THE FUTURE OF DRUG DISCOVERY: LIVE 3D CELL ANALYSIS

DF-BF Image

Total BF area x 105

+ BF Mask Outline

SK-BR-3

(μm2/image)
9

6

3
SK-BR-3
+ NHDF

+ NHDF

0

0

48

96
Time (h)

144

192

Total BF area x 105

Figure 2: Morphology observations with Incucyte® DF-BF images and quantification of
multi-spheroid size and kinetic growth using real-time analysis. SK-BR-3 cells were seeded
in flat bottom, 96-well plates on a bed of Matrigel in mono- or co-culture with NHDFs (1:1
ratio, 1,000 cells/well for each) and multi-spheroids (MS) allowed to form (3 d).

(μm2/image)
4

3

Incucyte®

extended depth of focus Brightfield (DF Brightfield) images (8 d post cell seeding) of

2

MCF7
MDA-MB-231
SK-BR-3
BT-474

SK-BR-3 MS in mono- or co-culture with NHDFs. Brightfield outline mask shown in yellow.
Note, the influence of NHDFs on SK-BR-3 MS morphology and size (Total Area). Time course
plots show the individual well Total Brightfield Object Area (μm2) (y-axis) over time (h)
(x-axis) and illustrate cell type specific kinetic growth profiles for a range of breast tumor
MS co-cultured with NHDFs. Data were collected over 192 h period at 6 h intervals. Each

1

0

0

data point represents mean ± SEM, n=15 wells.

48

96
Time (h)

144

192

pyruvate plus 1% non-essential amino acids and

gradient medium (StemCell Technologies) and

grown to confluence in 75 cm2 tissue culture

maintained in RPMI 1640 supplemented with

treated flasks (Corning). Cells were harvested and

10% heat inactivated FBS.

seeded into 96-well plates (Corning No. 3595)
coated with a base layer of 40 μL/well Matrigel

Influence of Stromal Cells on

such that 3 d post cell seeding, multi-spheroids

Multi-Spheroid Morphology

formed with desired size. Spheroid formation

NHDFs were shown to have a striking effect on

was monitored in an Incucyte® S3 over 3 d at 6

tumor multi-spheroid morphology. SK-BR-3 cells

h intervals. Peripheral blood mononuclear cells

were seeded alone or with NHDFs (1:1 ratio,

(PBMCs) were isolated from buffy coats from

1,000 cells/well for each) and DF Brightfield

healthy donors using Lymphoprep™ density

images acquired every 6 h. In mono-culture,

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Sartorius eBook

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