Erwin Berthier

Flow rate analysis of a surface tension driven passive micropump

A microfluidic passive pumping method relying on surface tension properties is investigated and a physical model is developed. When a small inlet drop is placed on the entrance of a microfluidic channel it creates more pressure than a large output drop at the channel exit, causing fluid flow. The behavior of the input drop occurs in two characteristic phases. An analytical solution is proposed and verified by experimental results. We find that during the first phase the flow rate is stable and that this phase can be prolonged by refilling the inlet drop to produce continuous flow in the microchannel.

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.

Managing evaporation for more robust microscale assays Part 1. Volume loss in high throughput assays

Evaporation is a well known issue when handling small liquid volumes. Here we present a review of microscale assays prone to evaporation and methods to make them more robust. Applications for these assays span from combinatorial chemistry to cell-biology where the stability of concentrations and osmolarity can be critical. A dimensionless evaporation number Ev is presented and used to characterize volume loss in short term and long term microscale assays. Ev can be used both as a design tool and as an analysis parameter. The advantageous use of evaporation in some applications is also discussed.

Suspended microfluidics

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

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.

Managing evaporation for more robust microscale assays Part 2.† Characterization of convection and diffusion for cell biology‡

Cell based microassays allow the screening of a multitude of culture conditions in parallel, which can be used for various applications from drug screening to fundamental cell biology research. Tubeless microfluidic devices based on passive pumping are a step towards accessible high throughput microassays, however they are vulnerable to evaporation. In addition to volume loss, evaporation can lead to the generation of small flows. Here, we focus on issues of convection and diffusion for cell culture in microchannels and particularly the transport of soluble factors secreted by cells. We find that even for humidity levels as high as 95%, convection in a passive pumping channel can significantly alter distributions of these factors and that appropriate system design can prevent convection.

Characterizing asthma from a drop of blood using neutrophil chemotaxis

Significance Asthma is a chronic inflammatory disorder that is notoriously difficult to diagnose, characterize, and properly treat with tests that are available to clinicians today. Therefore, clinicians would benefit from additional tools that objectively characterize asthma in patients. In this work, we describe a handheld microfluidic device that discriminates asthma from allergic rhinitis patients based on neutrophil function—an inflammatory cell that has been implicated in the pathogenesis of asthma. The device can sort neutrophils from a drop of whole blood within minutes, and was used in a clinical setting to characterize asthmatic and nonasthmatic patients. This technology provides a new tool for clinicians to characterize asthma based on cellular function. Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient’s neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.

Low-Volume Toolbox for the Discovery of Immunosuppressive Fungal Secondary Metabolites

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, a...