Edmond W. K. Young

Fundamentals of microfluidic cell culture in controlled microenvironments.

Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology.

Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels{{

The interactions between endothelial cells and the underlying extracellular matrix regulate adhesion and cellular responses to microenvironmental stimuli, including flow-induced shear stress. In this study, we investigated the adhesion properties of primary porcine aortic endothelial cells (PAECs) and valve endothelial cells (PAVECs) in a microfluidic network. Taking advantage of the parallel arrangement of the microchannels, we compared adhesion of PAECs and PAVECs to fibronectin and type I collagen, two prominent extracellular matrix proteins, over a broad range of concentrations. Cell spreading was measured morphologically, based on cytoplasmic staining with a vital dye, while adhesion strength was characterized by the number of cells attached after application of shear stresses of 11, 110, and 220 dyn cm(-2). Results showed that PAVECs were more well spread on fibronectin than on type I collagen (P < 0.0001), particularly for coating concentrations of 100, 200, and 500 microg mL(-1). PAVECs also withstood shear significantly better on fibronectin than on collagen for 500 microg mL(-1). PAECs were more well spread on collagen compared to PAVECs (P < 0.0001), but did not have significantly better adhesion strength. These results demonstrate that cell adhesion is both cell-type and matrix dependent. Furthermore, they reveal important phenotypic differences between vascular and valvular endothelium, with implications for endothelial mechanobiology and the design of microdevices and engineered tissues.

Rapid Prototyping of Arrayed Microfluidic Systems in Polystyrene for Cell-Based Assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

Simultaneous generation of droplets with different dimensions in parallel integrated microfluidic droplet generators

This paper describes geometric coupling of the dynamics of break-up of liquid threads in parallel flow-focusing devices (FFD), which are integrated into a multiple quadruple-microfluidic droplet generator (QDG). We show weak parametric coupling between parallel FFDs with an identical design, which leads to the slight broadening of the distribution of sizes of droplets. Using parallel FFDs with distinct geometries we simultaneously generated several populations of droplets with different volumes, yet, each of these populations was characterized by a narrow size distribution. Simulation of the generation of droplets in the quadruple-microfluidic droplet generator based on hydraulic resistances to the flow of a single-phase fluid was in good agreement with the experimental results.

Technique for Real-Time Measurements of Endothelial Permeability in a Microfluidic Membrane Chip Using Laser-Induced Fluorescence Detection

Characterizing permeability of the endothelium that lines blood vessels and heart valves provides fundamental physiological information and is required to evaluate uptake of drugs and other biomolecules. However, current techniques used to measure permeability, such as Transwell insert assays, do not account for the recognized effects of fluid flow-induced shear stress on endothelial permeability or are inherently low-throughput. Here we report a novel on-chip technique in a two-layer membrane-based microfluidic platform to measure real-time permeability of endothelial cell monolayers on porous membranes. Bovine serum albumin (a model protein) conjugated with fluorescein isothiocyanate was delivered to an upper microchannel by pressure-driven flow and was forced to permeate a poly(ethylene terephthalate) membrane into a lower microchannel, where it was detected by laser-induced fluorescence. The concentration of the permeate at the point of detection varied with channel flow rates in agreement to less than 1% with theoretical analyses using a pore flow model. On the basis of the model, a sequential flow rate stepping scheme was developed and applied to obtain the permeability of cell-free and cell-bound membrane layers. This technique is a highly sensitive, novel microfluidic approach for measuring endothelial permeability in vitro, and the use of micrometer-sized channels offers the potential for parallelization and increased throughput compared to conventional shear-based permeability measurement methods.

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

Microfluidic Cell Culture and Its Application in High-Throughput Drug Screening: Cardiotoxicity Assay for hERG Channels

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound’s risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.

Methylglyoxal-modified collagen promotes myofibroblast differentiation.

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p<0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p<0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.

Methylglyoxal Inhibits the Binding Step of Collagen Phagocytosis

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical α2β1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.

Microscale functional cytomics for studying hematologic cancers.

An important problem in translational cancer research is our limited ability to functionally characterize behaviors of primary patient cancer cells and associated stromal cell types, and relate mechanistic understanding to therapy selection. Functional analyses of primary samples face at least 3 major challenges: limited availability of primary samples for testing, paucity of functional information extracted from samples, and lack of functional methods accessible to many researchers. We developed a microscale cell culture platform that overcomes these limitations, especially for hematologic cancers. A key feature of the platform is the ability to compartmentalize small populations of adherent and nonadherent cells in controlled microenvironments that can better reflect physiological conditions and enable cell-cell interaction studies. Custom image analysis was developed to measure cell viability and protein subcellular localizations in single cells to provide insights into heterogeneity of cellular responses. We validated our platform by assessing viability and nuclear translocations of NF-κB and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-κB activation in a primary chronic lymphocytic leukemia patient sample. Our platform can be applied to myriad biological questions, enabling high-content functional cytomics of primary hematologic malignancies.