Fly Me to the Micron: Microtechnologies for Drosophila Research.

Multicellular model organisms, such as Drosophila melanogaster (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise. We discuss recent advances in microtechnological systems developed for small model organisms using D. melanogaster as an example. We critically analyze the state of the field by comparing the systems produced for different applications. Additionally, we suggest design guidelines, operational tips, and new research directions based on the technical and knowledge gaps in the literature. This review aims to foster interdisciplinary work by helping engineers to familiarize themselves with model organisms while presenting the most recent advances in microengineering strategies to biologists.

Polycarbonate Heat Molding for Soft Lithography.

Soft lithography enables rapid microfabrication of many types of microsystems by replica molding elastomers into master molds. However, master molds can be very costly, hard to fabricate, vulnerable to damage, and have limited casting life. Here, an approach for the multiplication of master molds into monolithic thermoplastic sheets for further soft lithographic fabrication is introduced. The technique is tested with master molds fabricated through photolithography, mechanical micromilling as well as 3D printing, and the results are demonstrated. Microstructures with submicron feature sizes and high aspect ratios are successfully copied. The copying fidelity of the technique is quantitatively characterized and the microfluidic devices fabricated through this technique are functionally tested. This approach is also used to combine different master molds with up to 19 unique geometries into a single monolithic copy mold in a single step displaying the effectiveness of the copying technique over a large footprint area to scale up the microfabrication. This microfabrication technique can be performed outside the cleanroom without using any sophisticated equipment, suggesting a simple way for high-throughput rigid monolithic mold fabrication that can be used in analytical chemistry studies, biomedical research, and microelectromechanical systems.

Microfluidics for understanding model organisms.

New microfluidic systems for whole organism analysis and experimentation are catalyzing biological breakthroughs across many fields, from human health to fundamental biology principles. This perspective discusses recent microfluidic tools to study intact model organisms to demonstrate the tremendous potential for these integrated approaches now and into the future. We describe these microsystems' technical features and highlight the unique advantages for precise manipulation in areas including immobilization, automated alignment, sorting, sensory, mechanical and chemical stimulation, and genetic and thermal perturbation. Our aim is to familiarize technologically focused researchers with microfluidics applications in biology research, while providing biologists an entrée to advanced microengineering techniques for model organisms.

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

Endothelial cell polarization and orientation to flow in a novel microfluidic multimodal shear stress generator.

Endothelial cells (EC) respond to shear stress to maintain vascular homeostasis, and a disrupted response is associated with cardiovascular diseases. To understand how different shear stress modalities affect EC morphology and behavior, we developed a microfluidic device that concurrently generates three different levels of uniform wall shear stress (WSS) and six different WSS gradients (WSSG). In this device, human umbilical vein endothelial cells (HUVECs) exhibited a rapid and robust response to WSS, with the relative positioning of the Golgi and nucleus transitioning from a non-polarized to polarized state in a WSS magnitude- and gradient-dependent manner. By contrast, polarized HUVECs oriented their Golgi and nucleus polarity to the flow vector in a WSS magnitude-dependent manner, with positive WSSG inhibiting and negative WSSG promoting upstream orientation. Having validated this device, this chip can now be used to dissect the mechanisms underlying EC responses to different WSS modalities, including shear stress gradients, and to investigate the influence of flow on a diverse range of cells during development, homeostasis and disease.

Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers.

Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.

High-throughput mechanotransduction in Drosophila embryos with mesofluidics.

Developing embryos create complexity by expressing genes to coordinate movement which generates mechanical force. An emerging theory is that mechanical force can also serve as an input signal to regulate developmental gene expression. Experimental methods to apply mechanical stimulation to whole embryos have been limited, mainly to aspiration, indentation, or moving a coverslip; these approaches stimulate only a few embryos at a time and require manual alignment. A powerful approach for automation is microfluidic devices, which can precisely manipulate hundreds of samples. However, using microfluidics to apply mechanical stimulation has been limited to small cellular systems, with fewer applications for larger scale whole embryos. We developed a mesofluidic device that applies the precision and automation of microfluidics to the Drosophila embryo: high-throughput automatic alignment, immobilization, compression, real-time imaging, and recovery of hundreds of live embryos. We then use twist:eGFP embryos to show that the mechanical induction of twist depends on the dose and duration of compression. This device allows us to quantify responses to compression, map the distribution of ectopic twist, and measure embryo stiffness. For building mesofluidic devices, we describe modifications on ultra-thick photolithography, derive an analytical model that predicts the deflection of sidewalls, and discuss parametric calibration. This "mesomechanics" approach combines the high-throughput automation and precision of microfluidics with the biological relevance of live embryos to examine mechanotransduction. These analytical models facilitate the design of future devices to process multicellular organisms such as larvae, organoids, and mesoscale tissue samples.