Multi-cellular tumor spheroids ͑MCTSs͒ have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is timeconsuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane ͑PDMS͒ microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.
We present a novel method to create cavities in PDMS that is simple and exhibits wide process latitude allowing control over the radius of curvature to form shallow concave pits or deep spherical cavities.Polydimethylsiloxane (PDMS) is a silicon-based polymer exhibiting tunable mechanical, chemical and optical properties that are widely leveraged in microfluidic and biotechnology applications. Its clear optical properties are especially advantageous for spectroscopic analysis through lab-on-a-chip devices. 1-4 Its compliant properties are exploited to perform large area lithography on non-planar surfaces 5 and to form variable-focus microlens arrays. 6-8 PDMS is inert and non-toxic, making it well suited for cell culture and tissue engineering applications. 9,10 PDMS stamps are used to spatially pattern antibodies, enzymes, protein ligands and cells onto sensors and other material surfaces using the micro-contact printing technique. 11-15 PDMS stamps are typically fabricated by reverse replication of features formed in a silicon wafer mold.The most widely used PDMS elastomer is Dow Corning Sylgard 184 (1 part catalyst : 10 parts base by weight) which is degassed prior to filling the mold. This material has a high elongation at break (160%) and a relatively low modulus (1.8 MPa). 16 Features molded in PDMS are typically rectilinear forms, including squares, rectangles, cylinders and composite geometries combining these. More complex shapes (spheres and ellipsoids) that are needed to form, for example, microlens arrays require innovative process development. Some recent examples include photoresist thermal reflow processing, 17,18 gel trapping, 8 mechanical deformation of elastomeric mold, 19 liquid-filled cavities 20 and surface tension extrusion. 21In this communication we report a new method to create smooth concave cavities in PDMS that allows control over the radius of curvature to form shallow pits or deep spherical cavities. While these structure are useful for microlens molding, our interest is to create microbioreactor arrays. Leveraging novel fluidic flows and affinity capture methods, cells can be sorted into spatially distinct reactors of different sizes, where they can be maintained on-chip for research purposes or until needed for therapeutic use. 10,22,23Our method, outlined in Fig. 1, utilizes standard lithographic processing to create a silicon wafer mold comprised of deep reactive ion etched (DRIE) trenches (Fig. 1A). An SEM image of a typical wafer mold containing linear arrays of DRIE trenches measuring 95 × 95 × 350 μm is illustrated in Fig. 2A. These trenches were fabricated using the Bosch process that NIH Public Access consists of sequential SF 6 etching and C 4 F 8 passivation steps. 24 The passivation step deposits a hydrophobic fluoropolymer coating that protects against side wall erosion as etching proceeds deeper into the silicon wafer. Normally, the hydrophobic passivation layer is removed prior to subsequent processing steps using oxygen plasma. Our method for molding cavitie...
The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, and disease development and progression. In this study we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 µm in diameter formed in hydrophobic polymer polydimethylsiloxane (PDMS) with ~60–100 µm circular openings and aspect ratio ~3.5. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells / MB) and cultured under standard conditions, adopt a compact 3-D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96 well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca2+ concentration and changes in Ecadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.
Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in(2)). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in(2) with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.
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