In summary, this work presented a microfluidic chip that can study the in vitro 3D tumor migration and the anti-cancer drug screening at the same chip. The chip is suitable for real time observation instead of endpoint assay and work as a tubeless system to facilitate the experimental process. We also demonstrate the potential of the chip for long term culture of the cells acquired metastasis ability which might be cancer-stem-like cells. In the future, the chip can be co-cultured like endothelial cell in EGF channel to better mimic blood vessel so that we can study the integrated metastasis process of tumor cell. Thus, the present device provides a viable approach to realize long term organotypic cultures that represents suitable tumor microenvironment, and may facilitate personalized drug testing.
Appendix
Biomimetic nano-cilia generate multicellular tumor spheroids
Figure 10a presents the methodology of 3D multicellular spheroid cultures that rely on engineered nano-cilia and cell-cell interactions, which is based on the steric repulsion of cell-to-substrate adhesion and subsequent locomotion and self-aggregation of cells into a tumor spheroid. In this study, we adopted triblock copolymer monolayer – Pluronic F108 – to as a candidate of such the biomimetic nano-cilia. Pluronic F108 is known for its nontoxicity and biocompatibility for a wide range of cellular study. It has two separated hydrophilic PEO chain lengths of 128 monomer units and a hydrophobic PPO chain length of 54 units, and the fully extending PEO chain length in solution would be approximately 45 nm, thereby it may be easily adjusted to typical cell culture platforms and micro-scale technology without interfering the microscopy observation.
In addition, we have recently demonstrated that the Pluronic copolymers may be applied for the deterministic 2D/3D cell patterning by microfluidics. However, the combination of biomimetic nano cilia-mediated repulsion, locomotion, and self-aggregation to achieve 3D spheroid cultures and the subsequent phenotypic/genotypic characteristics from the resulted spheroids have not yet been systematically studied.
To first evaluate and then confirm the feasibility of triblock copolymers – coated on conventional polystyrene (PS) dishes/plates – for spheroid cultures, we applied several methodology, including multiple cell lines testing for spheroid generation, contact-angle measurement, protein repulsion, immunofluorescence detection, and long-term viability testing (Figures 10b and c, Figure 11). Figure 10b presents the
contact angle images (top panel) and the proposed schematics of the chain alignment (bottom panel). The hydrophobic PPO chains were bound onto the PS surface due to hydrophobic-hydrophobic interaction, and then the flexible and hydrophilic PEO chains were swung freely (with a beating frequency of approximately 10 GHz) in the hydrated layer of solutions, suggesting that this resulted in a decreased contact angle of 64°
comparing to native PS of 77 ° . MCF7 breast cancer cells cultured in Pluronic copolymers (1% F108) formed multicellular spheroids, similar to those found in mammospheres except no supplementary mitogen used in this study, whereas cells cultured on traditional tissue culture dishes formed typical 2D monolayers (Figure 10c).
In addition, we prepared Pluronic F108 of different concentrations coated onto PS dishes and then determined that 1% F108 was the optimal polymer for multicellular aggregate cultures and steric repulsion of hydrophobic molecules/proteins (Figure 11).
Notably, results from Figure 2b indicate that PS’s hydrophobic nature tends to absorb small hydrophobic molecules in culture medium and might decrease the water contact angle as well as lower polymer coating (0.01% and 0.1% F108). In contrast, 1%
F108-coated PS wound efficiently prevent the absorption of molecules and proteins, thereby it has the capacity for 3D spheroid cultures to diminish cell-to-substrate adhesion. Similar to MCF7 and SKOV3 cells, human pancreatic cancer cell line (Panc 02.03B) and two human colorectal cancer cell lines (DLD-1 and SW480) all formed
multicellular spheroids in 1% F108 as well. The biomimetic nano-cilia by Pluronic F108 thus have potential to achieve modeling of tumor spheroids in vitro.
Figure 10 3D spheroid cultured with triblock-copolymer (nano-cilia)-based locomotion. a, Illustrations showing the configuration of the triblock copolymers (nano-cilia) system utilized for 3D multicellular spheroid cultures, in which x and y indicate the monomer units. Through the hydrophobic-hydrophobic interaction, the hydrophobic PPO chains will bind onto the PS surface and then beat the hydrophilic PEO chains freely in medium, thereby diminishing the cell-to-substrate adhesion and directing cell-cell interactions to organize a cellular spheroid. b, DI water drop with blue dye on PS surface before and after treatment with copolymers (1% Pluronic F108).
A contact angle change of 13° was observed. c, Morphology of MCF7 cells cultured in conventional 2D monolayers and 3D spheroids as shown by phase contrast (top panel) and immunofluorescence (bottom panel; cell adhesion molecule stained with EpCAM, nuclear with Hoechst) images. Scale bar, 100 µm.
Figure 11 Characteristics of triblock copolymers in steric repulsion. (a) SKOV3 monolayer was cultured in collagen gel (100 µg/ml)-coated PS surface (control), whereas multicellular aggregates were generated after one day culture in triblock copolymers of different concentrations (experiment). Scale bar, 100 µm. (b) Comparison of contact angles on native and copolymer-coated PS surfaces. Colors in blue and red represent the surfaces without any treatment and by dipping in cell culture medium for 1 h, respectively. Contact angles were measured by dripping of a 1 µL droplet of DI water on the PS surfaces. The data are presented as mean ± SD from three independent experiments (*** p < 0.001).
Experimental methods to study tumor cell migration
Micropipette assay: a pipette is placed in the vicinity of the cell and a chemoattractant
solution is injected into the culture medium establishing a growth-factor gradient.
Boyden (or Trans-well) chamber : cells are seeded in suspension in the top chamber
and migrate through the porous filter in response to a chemokine gradient, which is established by the different culture medium concentrations in the top and bottom chambers.
Micropatterning: cells are seeded on patterns of different geometry, size and surface
coatings and their migration characteristics are monitored.
Durotaxis: cells are seeded on a substrate of variable stiffness and respond by changing
traction forces, cell spread area, and migration direction.
Wound healing: a ‘‘wound’’ is formed on a confluent tumor monolayer, and the wound closure dynamics are monitored.
3D ECM: cells are seeded inside the 3D ECM and migrate depending on the ECM
architecture (stiffness, pore size, and ligand concentration); ECM fibers are outlined with black curved lines.
Microfluidics: cytokine gradients can be established in a 3D matrix by flowing different
chemokine concentration solutions in the left and right microchannels; Interstitial flow can be established by adjusting the hydrostatic pressure in the left and right
microchannels; streamlines are indicated with dark magenta lines. Micropipette, Boyden chamber and microfluidics assays enable control of biochemical gradients. Durotaxis, 3D ECM and microfluidics assays enable control of biophysical forces (ECM stiffness
and interstitial flow). Wound healing and micropatterning assay enable control of intercellular distances, whereas only micropatterning assays enable
control of substrate topography. The schematics of each method are showed in Fig. 12, and the advantages/limitations of each method are summarized in Table 1.
Figure 12 Experimental methods for investigating factors that influence tumor cell migration.
Table 1 Comparison of in vitro experimental approaches to study tumor cell migration
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