Co-culture of Two Different Cell Lines in a Two-Layer Microfluidic Device.

Microfluidic devices have become a promising alternative approach for cellular co-culture. Many approaches incorporate a semipermeable barrier to physically separate, yet chemically connect, two cell types; however, the majority of these approaches utilize batch culture conditions which can result in nutrient depletion and waste accumulation. This chapter describes an alternative approach that allows for the continuous infusion of media, relieving the constraints of batch culture. The microfluidic device consists of two separate layers: a bottom layer of 3% (w/v) agarose to facilitate chemical diffusion and a top polydimethylsiloxane (PDMS) layer into which four parallel fluidic channels were imprinted. The microfluidic approach allows for facile visualization of cells with light microscopy and the ability to add (or subtract) drugs or biomolecules to interrogate the system or modulate the cellular response. Finally, the approach allows for terminal immunostaining of either (or both) cell types.

Evaluation of intercellular communication between breast cancer cells and adipose-derived stem cells via passive diffusion in a two-layer microfluidic device.

Breast cancer tumorigenesis and response to therapy is regulated by cancer cell interactions with the tumor microenvironment (TME). Breast cancer signaling to the surrounding TME results in a heterogeneous and diverse tumor microenvironment, which includes the production of cancer-associated fibroblasts, macrophages, adipocytes, and stem cells. The secretory profile of these cancer-associated cell types results in elevated chemokines and growth factors that promote cell survival and proliferation within the tumor. Current co-culture approaches mostly rely on transwell chambers to study intercellular signaling between adipose-derived stem cells (ASCs) and cancer cells; however, these methods are limited to endpoint measurements and lack dynamic control. In this study, a 4-channel, "flow-free" microfluidic device was developed to co-culture triple-negative MDA-MB-231 breast cancer cells and ASCs to study intercellular communication between two distinct cell types found in the TME. The device consists of two layers: a top PDMS layer with four imprinted channels coupled with a bottom agarose slab enclosed in a Plexiglas chamber. For dynamic co-culture, the device geometry contained two centered, flow-free channels, which were supplied with media from two outer flow channels via orthogonal diffusion through the agarose. Continuous fresh media was provided to the cell culture channel via passive diffusion without creating any shearing effect on the cells. The device geometry also allowed for the passive diffusion of cytokines and growth factors between the two cell types cultured in parallel channels to initiate cell-to-cell crosstalk. The device was used to show that MDA-MB-231 cells co-cultured with ASCs exhibited enhanced growth, a more aggressive morphology, and polarization toward the ASCs. The MDA-MB-231 cells were found to exhibit a greater degree of resistance to the drug paclitaxel when co-cultured with ASCs when compared to single culture studies. This microfluidic device is an ideal platform to study intercellular communication for many types of cells during co-culture experiments and allows for new investigations into stromal cell-mediated drug resistance in the tumor microenvironment.

Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device.

Even though the majority of breast cancers respond well to primary therapy, a large percentage of patients relapse with metastatic disease, for which there is no treatment. In metastasis, a tumor sheds a small number of cancerous cells, termed circulating tumor cells (CTCs), into the local vasculature, from where they spread throughout the body to form new tumors. As CTCs move through the circulatory system, they experience physiological forces not present in the initial tumor environment, namely, fluid shear stress (FSS). Evidence suggests that CTCs respond to FSS by adopting a more aggressive phenotype; however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies involves inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast cancer cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of multiple breast cancer types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing duration and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5 min of FSS, while MDA-MB-231 cells were observed to deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of distinct cell subpopulations with different phenotypes.

Microfluidic and Paper-Based Devices for Disease Detection and Diagnostic Research.

Recent developments in microfluidic devices, nanoparticle chemistry, fluorescent microscopy, and biochemical techniques such as genetic identification and antibody capture have provided easier and more sensitive platforms for detecting and diagnosing diseases as well as providing new fundamental insight into disease progression. These advancements have led to the development of new technology and assays capable of easy and early detection of pathogenicity as well as the enhancement of the drug discovery and development pipeline. While some studies have focused on treatment, many of these technologies have found initial success in laboratories as a precursor for clinical applications. This review highlights the current and future progress of microfluidic techniques geared toward the timely and inexpensive diagnosis of disease including technologies aimed at high-throughput single cell analysis for drug development. It also summarizes novel microfluidic approaches to characterize fundamental cellular behavior and heterogeneity.