Supplementary Information for

Configurable 2D and 3D Spheroid Tissue Cultures on

Bioengineered Surfaces with Acquisition of Epithelial-

Mesenchymal Transition Characteristics

Ching-Te Kuo1, Chi-Ling Chiang2, Ruby Yun-Ju Huang3,4, Hsinyu Lee2, and Andrew M. Wo1*

1Institute of Applied Mechanics, National Taiwan University, Taipei, Taiwan2Department of Life Science, National Taiwan University, Taipei, Taiwan

3Department of Obstetrics & Gynaecology, National University Hospital, Singapore4Cancer Science Institute of Singapore, National University of Singapore, Singapore

*E-mail: andrew@iam.ntu.edu.tw

Supplementary information files include a word file and a movie file. The word file

includes seven figures, an instruction for the movie, and a reference.

Supplementary Figures

Figure S1 Comparison of the contact angles on native PDMS and Pluronic copolymers

(1% F108)-coated PDMS surfaces. Colors in blue and red represent the surfaces without

any treatment and by dipping in cell culture medium for one hour, respectively. Contact

angles were measured by dripping of a 1 L droplet of deionized water on the PDMS

surfaces. Each test was repeated in three times. Results showed the wetting behavior of the

two copolymers-coated surfaces does not change, however, it’s contact angle of the native

PDMS surface dipping in medium changes closely to zero degree (i.e. from hydrophobic

to hydrophilic). These indicate that PDMS’s hydrophobic nature tends to absorb small

hydrophobic molecules in culture medium1 and might decrease the contact angle. In

contrast, the copolymers-coated PDMS will prevent the absorption of molecules or

proteins, and thus, it has the potential for cell patterning to prevent cell attachment.

Figure S2 Configurable cell patterning. Cells can be arranged into different shapes based

on the arrangement of through holes, such as (a) a symbol “L” pattern, (b) a hexagonal

pattern with a diagonal width of 70 m, and (c) a line pattern with width down to the

single-cell dimension.

Figure S3 Long-term cell viability in the microfluidic chip. Here we present the evidence

that the trapped cells (SKOV3 cells) cultured in the microchip were viable and can

proliferate for 6 days. Less numbers of cells (~ 5 cells trapped onto the circular pattern)

were used, and the cells were then monitored over a period of 6 days, as shown by

photographs (a-d) which show the exact same location over time. These results show that

cells can elongate and proliferate on the local patterned surface over 6 days and also

indicate that cells are viable during the time period. Scale bar, 100 m.

Figure S4 3D culture on the Pluronic copolymers-coated Petri-dish. (a) Schematics of

methodology (see the MATERIALS AND METHODS section). Suspended single cells

were plated to a copolymers (1% F108)-coated dish, and these cells will self-aggregate

into multicellular spheroids and be observed by microscope every day. (b) Photography of

the SKOV3 spheroids for 5 days after the onset of plating. The black arrows indicate the

location of the spheroids. Spheroids were counted and used to calculate the mean diameter,

in which we only counted the spheroids clustered at least by two cells. Scale bar, 100 m.

Figure S5 3D on-chip spheroid culture generates EMT properties more than that in 3D on-

dish culture. The expression levels of the mRNAs encoding E-cadherin, N-cadherin,

vimentin, and fibronectin in cells derived from 3D on-chip spheroid growth relative to 3D

on-dish culture, as determined by real-time RT-PCR. GAPDH was used to normalize the

variability in sample loading. The data were presented as mean ± SEM from three

independent experiments.

Figure S6 Cells off chip induces mesenchymal to epithelial reverting transition (MET).

Cells on chip represents the cells from the primary spheroids, cultured on chip for 5 days.

Cells off chip represents the cells derived from the chip, and further cultured in

conventional 2D manner for 12 days. The expression levels of the mRNAs encoding E-

cadherin, N-cadherin, vimentin, and fibronectin in cells derived from off-chip growth

relative to on-chip growth, as determined by real-time RT-PCR. GAPDH was used to

normalize the variability in sample loading. The data were presented as mean ± SEM

from three independent experiments.

Figure S7 In situ modeling of migration of tumor cells in 3D matrices. (a) A schematic

showing the experimental design: (1) a tumor spheroid first grows onto the membrane

surface; (2) 3D ECM scaffolding within the microchip. Meanwhile, cells will undergo

EMT due to the reactive microenvironment; (3) these cells may acquire metastatic

potential and migrate to a distant site. This design may enable for some applications, for

example, solvable factors or chemo-drugs can be introduced into the microchip from the

bottom channel to screen for a more suitable therapy for metastatic cancer diseases.

BV/LV, blood vessels/lymphatic vessels. (b) Results show the migration of tumor cells in

3D in the microfluidic chip. When the cellular spheroid grew in the chip at day 6, collagen

gel (1 mg/ml in PBS) was introduced to the top channel as a scaffold material and gelled

in an incubator at 37°C for one hour. Following gelation, cell culture medium was then

introduced to whole channels and the chip was placed in an incubator at 37°C overnight.

At day 7, bright-field images were captured and results show some cells migrated to a

distant site (~ 200 m far) from the primary spheroid. Scale bar, 100 m.

Supplementary Movie

Movie S1 Top view of SKOV3 cancer cells being trapped onto the patterned porous

surface. 100 L of SKOV3 cells (~1.5 × 103 cells) in culture medium were introduced

into the top channel by adjusting a water height (Δh_top = 1 mm); meanwhile, a solution

height (Δh_bottom = 4 mm) was produced to create a suction to trap cells onto the

microfabricated holes. The movie was recorded with a CCD camera at 30 fps for 10

minutes.

Reference

1. Hsiung, L.-C., Chiang, C.-L., Wang, C.-H., Huang, Y.-H., Kuo, C.-T., Cheng, J.-Y., Lin, C.-H., Wu, V., Chou, H.-Y., Jong, D.-S., Lee, H. & Wo, A. M. Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments. Lab on a Chip 11, 2333-2342 (2011).