In vitro assays for determining the metastatic potential of melanoma cell lines with characterized in vivo invasiveness.

The metastatic potential of cancer cells is an elusive property that is indicative of the later stages of cancer progression. The ability to distinguish between poorly and highly metastatic cells is invaluable for understanding the basic biology of cancer and to develop more treatments. In this paper, we exploit a A375 melanoma cell line series (A375P, A375MA1, A375MA2) that vary in metastatic potential, to demonstrate an in vitro screening assay using polydimethylsiloxane (PDMS) microbubble well arrays that can distinguish these cell lines by their growth characteristics in including morphology, migratory potential, and clonogenic potential. These cell lines cannot be distinguished by their growth characteristics when cultured on standard tissue culture plastic or planar PDMS. Results show that the more metastatic cell lines (A375MA1, A375MA2) have a higher proliferative potential and a distinctive radial spreading growth pattern out of the microbubble well. The A375MA2 cell line also has a higher tendency to form multicellular spheroids. The ability to successfully correlate the metastatic potential of cancer cells with their growth characteristics is essential first step toward developing a high-throughput screening assay to identify aggressive tumor cells in primary samples. The capability to culture and recover aggressive cells from microbubble wells will enable identification of candidate metastatic biomarkers which has immense clinical significance.

Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications.

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.

Microenvironment induced spheroid to sheeting transition of immortalized human keratinocytes (HaCaT) cultured in microbubbles formed in polydimethylsiloxane.

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, 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 polydimethylsiloxane (PDMS) with ∼60-100 µm circular openings and aspect ratio ∼3.0. 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 3D 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 undergo a 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-ß1 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-ß1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca(2+) concentration and changes in E-cadherin 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.

Continuously perfused microbubble array for 3D tumor spheroid model.

Multi-cellular tumor spheroids (MCTSs) 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 time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) 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.

Microfabrication of cavities in polydimethylsiloxane using DRIE silicon molds.

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.