Fig 1.
Schematic illustration of the study strategy.
For each cell line used, 3D spheroid formation was evaluated by four different methods, i.e., PDMS-based microfluidic systems, ultra-low attachment (ULA) plates in the presence or not of 2% Matrigel, and hanging droplets. The two most effective 3D spheroid methods (microfluidic chips and Matrigel-assisted ULA plates) were used to define carboplatin sensitivity of each cell line that was then compared to responses in the 2D monolayer assay.
Table 1.
Carboplatin sensitivity (IC50 by clonogenic assay) of the EOC cell lines used in this study.
Fig 2.
Design of PDMS microfluidic device developed for the culture of 3D spheroids and drug testing.
Each chip is comprised of five sections of 24 wells, containing 120 wells. Each well has a dimension of 500×500×500 μm3. The scale bar is 1 cm.
Fig 3.
Capacity of EOC cell lines to form spheroids in three different culture conditions.
Representative microscopic bright field images of TOV3041G, TOV112D, OV90 and OV866(2) cells cultured 48 hours in hanging droplets (A), within the custom PDMS-based microfluidic chips, (B) and in 96-well round-bottom ULA plates with 2% Matrigel (C). The scale bar is 500 μm.
Fig 4.
Carboplatin response of EOC spheroids cultured in PDMS microfluidic devices.
3D spheroids of four EOC cell lines [TOV3041G, TOV112D, OV90, OV866(2)] were formed and cultured in microfluidic devices for 2 and 4 days (Ctrl Day 2 and Ctrl Day 4) or treated at day 2 with 300 μM of carboplatin and assessed at day 4 (Carbo 300 Day 4). A) Representative microscopic bright field images at 10X magnification. B) Comparison of carboplatin treatment on EOC cell survival (grey bars) and spheroid diameters (red points). 48 spheroids were analyzed (in duplicate) from each set of experiment readouts. Error bars indicate the standard errors of the mean (SEM) of three independent experiments. Statistical analyses: Ctrl Day 4 vs Carbo 300 Day 4, * p<0.05; *** p<0.0005; ns, non-significant (Student’s t-test).
Fig 5.
Carboplatin response of EOC spheroids cultured with 2% matrigel in ultra-low attachment wells.
3D spheroids of TOV3041G (A), TOV112D (B), OV90 (C) and OV866(2) (D) were formed and cultured in concave-bottom ULA plates in the presence of 2% Matrigel for 4 days (Ctrl Day 4) or treated at day 2 with 300 μM of carboplatin and assessed at day 4 (Carbo 300 Day 4). Top panels are representative microscopic bright field images of each condition. Bar graphs represent cell survival analysed by flow cytometry of control (light grey) and treated (dark grey) spheroids. Data are shown as the mean ± SEM of three independent experiments. * p<0.05; ** p<0.005 (Student’s t-test).
Fig 6.
Comparison of carboplatin response between 2D-monolayers and two different 3D spheroid models.
A) TOV3041G, TOV112D, OV90 and OV866(2) were culture as 2D monolayers for 4 days without treatment (Ctrl Day 4) or treated at day 2 with 300 μM of carboplatin and assessed at day 4 (Carbo 300 Day 4). Bar graphs represent cell survival analysed by flow cytometry of control (light grey) and treated (dark grey). Data are shown as the mean ± SEM of three independent experiments * p<0.05; ** p<0.005; ns, non-significant (Student’s t-test) B) 2D-monolayer carboplatin sensitivity was compared to that of 3D spheroid cultures. Each treatment response was normalized relative to the percentage of live cells in comparison to the appropriate control. * p-value <0.05; ** p-value <0.005; *** p-value <0.0005; Turkey ANOVA multi comparison test of three independent experiments.
Table 2.
Relative carboplatin sensitivity of four EOC cell lines in three different culture conditions.