DUAL ADHERENT/SUSPENSION CO-CULTURE MICRO-ENVIRONMENT FOR THE STUDY OF CANCER-STROMAL

INTERACTIONS USING SINGLE-CELL DERIVED SPHERE FORMATION

Yu-Chih Chen1, Zhixiong Zhang1 and Euisik Yoon1, 2 1Dept. of Electrical Eng. and Computer Science, University of Michigan, Ann Arbor, MI, USA

2Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

ABSTRACT Considerable evidence suggests that cancer-stromal interactions are critical in tumor pathogenesis,

but conventional dish-based co-culture methods lack in capability of precisely controlling micro-environments and tracking single cell behavior. To better model tumorigenesis, we present a dual adher-ent/suspension co-culture device, which can provide both a suspension environment for cancer cells and an adherent environment for stromal cells in close proximity by selectively patterning polyHEMA in in-dented micro-wells. For investigating cellular heterogeneity, single tumor cells were monitored in a mi-crowell surrounded by stromal cells. As the proof of concept, we have successfully demonstrated the ele-vated stemness and proliferation rate induced by cancer-stromal interactions.

KEYWORDS: Co-Culture, Cancer, Single Cell, Tumor Sphere, Cell-Cell Interaction

INTRODUCTION

Cancer-stromal interactions are critical in tumorigenesis, but conventional dish-based co-culture methods lack in capability of precisely controlling micro-environments and tracking single cell behavior [1-3]. To understand the heterogeneity of cancer cells, ideal co-culture platforms should be able to pro-vide single-cell resolution for characterizing individual cell behaviors rather than average behavior [4]. There are a number of previous works reporting on cell-cell interaction with single-cell resolution [5-8], but those works are limited to adherent co-culture. The 3D suspension culture is believed to be better in modelling tumorigenesis, while suspension environment is too harsh for most adherent stromal cells (e.g., fibroblast cells) to survive [9-10]. To the best of our knowledge, this paper reports the first co-culture platform combining both suspension and adhesion cultures in close proximity. In addition, the presented platform allows the use of small samples (<100 cells), granting a potential to study rare cells such as CTCs.

EXPERIMENTAL

The presented co-culture platform consists of an inner suspension-culture chamber and an outer ad-herent-culture chamber connected by narrow (3µm height) interaction channels, which limit cell migra-tion but allow paracrine-based interactions (Fig. 1). We developed fabrication processes to selectively pattern polyHEMA, a non-adherent polymer [11], inside the inner chambers to prevent cell attachment for suspension culture. We poured polyHEMA on the patterned PDMS substrate in ethanol solution, removed excessive polyHEMA solution outside the patterned microwells by squeezing the cap [11], and evapo-rated ethanol to deposit polyHEMA selectively in the microwells for suspension culture. The fabricated substrate is treated by oxygen plasma to remove polyHEMA residues and then aligned and bonded to an-other PDMS fluidic layer which forms outer chambers and interaction channels. The SEM picture (Fig. 2(B)) shows polyHEMA filled only in a micro-well, while the top surface remains uncoated. The profile of the surface was shown in Fig. 2(C, D). T47D (breast cancer) cells were placed on the fabricated sub-strate, demonstrating suspension culture in the microwell and adherent culture on the surrounding areas (Fig.3(D)).

305978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

Fig.1. microfluidic chip for adhesion/suspension co-culture: (A) a fabricated device having 120 chambers, and (B) a fabricated co-culture chamber (scale bar: 100 µm).

Fig.2. Fabrication of non-adherent microwell: (A, B) SEM of a microwell before and after filling polyHEMA and (C, D) surface profile of a microwell before and after filling polyHEMA measured by LEXT. PolyHEMA was measured to be 4 µm thick in the center. (scale bar: 100 µm)

RESULTS AND DISCUSSION In our experiments, stromal cells are first loaded to outer chambers (Fig. 4(B)) and cultured for 2 days to form a monolayer (Fig. 4(C)). Single T47D cells are then loaded to inner chambers (Fig. 4(D), Fig. 5(A)). Among 50 loaded cells, 37 single cells were isolated in the chambers, as predicted by Poisson distribution (Fig. 5(B)). After co-cultured for 14 days, we counted the number of single-cell-derived spheres [11-12]. Compared to mono-cultured T47D cells (Fig. 6(A)), the co-cultured cells with cancer associated fibro-blast (CAF) (Fig. 6(B)) has a higher sphere formation rate by three times (Fig. 6(C)). Co-cultured spheres were also observed larger (Fig. 6(D)), indicating co-culture with CAF enhances sphere formation (stem-ness) as well as proliferation of cancer cells.

Fig.3. T47D cells grow on: (A) Petri dish (all adherent), (B) micro-well without coating (all adherent), (C) surface coated with polyHEMA (all suspension) and (D) substrate selectively coated with polyHEMA (suspension in the microwell but adherent elsewhere). (scale bar: 100 µm)

Fig.4. Cell loading process: (A) schematics of the a co-culture chamber, (B) stromal cells loaded in the outer culture chamber on day 0, (C) stromal cells adheres and grows to a monolayer on day 2, and (D) single cancer cell loaded in the inner chamber by revsersing the flow.

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Fig.5. (A) Co-culturing of adherent stromal cells and suspended single cancer cell and (B) distribution of the number of captured cancer cells per chamber when loading 50 cells into a 120-well device. (scale bar: 100 µm)

Fig.6. Cancer associated fibroblast boosts the sphere formation of T47D cancer cells: Representative cancer spheres on day 14 from (A) mono-cultured or (B) co-cultured. (C) Sphere formation rate of mono-cultured vs. co-cultured T47D cells after 14-day. (N = 5), ** P < 0.01. (D) Average size of mono-cultured and co-cultured T47D cells (N = 5), * P < 0.05. (scale bar: 100 µm)

CONCLUSION

We have successfully demonstrated a dual adherent-suspension co-culture platform, implemented by selectively patterning of polyHEMA in indented PDMS micro-wells. The suspension culture is ideal for investigating the tumorgenesis of cancer cells, and the adherent part is favorable for the survival of stromal cells. Using this platform, the elevated stemness and proliferation rate of T47D cells caused by cancer-stromal interactions was demonstrated. ACKNOWLEDGEMENTS

This work was supported in part by the Department of Defense (W81XWH-12-1-0325) and in part by the National Institute of Health (1R21CA17585701), and the CAF cells were provided by the Dr. Max Wicha's group.

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