Microfluidic perfusion system for culturing and imaging yeast cell microarrays and rapidly exchanging media

Abstract

High resolution live cell microscopy is increasingly used to detect cellular dynamics in response to drugs and chemicals, but it depends on complex and expensive liquid handling devices that have limited its wider adoption. Here, we present a microfluidic perfusion system that is built without using specialized microfabrication infrastructure, simple to use because only a pipette is needed for liquid handling, and yet allows for rapid media exchange and simultaneous fluorescence microscopy imaging. Yeast cells may be introduced from a culture, or spotted as arrays on a coverslip, and are sandwiched with a 20 mum thick track-etched membrane. A second coverslip and a mesh with 120 mum porosity are placed on top, forming a microfluidic conduit for lateral flow of solutions by capillary effects. Solutions introduced through the inlet flow through the mesh and chemicals diffuse vertically across the membrane to the cells trapped below. Solutions are exchanged by adding a new sample to the inlet. Using this system, we studied the dynamic response of F-actin in living yeast expressing Sac6-EGFP-a protein associated with discrete F-actin structures called "patches"-to the drug latrunculin A, a well known inhibitor of actin polymerization. We observed that the patches disappeared in 85% of the cells within 5 min, and re-assembled in 45 min following exchange of the drug with media. The perfusion system presented here is a simple, inexpensive device suited for analysis of drug dose-response and regeneration of single cells and arrays of cells.

Capillary pumps with constant flow rate

“…Note that in this example, the design features a CP consisting of parallel channels with nearly constant rectangular cross--section. This is possible because the flow resistance of the CP << R0, which was the a priori design requirement of previous CP designs (Eddowes, 1988;Juncker, 2002;Gervais, 2011;Mirzaei, 2010). Capillary pumps are often designed as micropillar arrays, which, due to their large amount of fluidic interconnections, are inherently robust against channel blockage by debris and against manufacturing imperfections.…”

“…For increasing positions s, Rh(s) increases. Consequently, a constant flow design requires a capillary suction P(s) that increases with s. CP designs with a constant porosity, and hence constant capillary pressure (Eddowes, 1988;Juncker, 2002;Gervais, 2011;Mirzaei, 2010) The maximum channel width occurs at s = 0, where w(0) = wmax is a design parameter. A decreasing w(s), in combination with a hydrophylic material surface with equilibrium contact angle < !…”

“…Previous CP design approaches for providing a constant flow are based on providing a constant capillary pressure throughout the CP while not significantly increasing the overall flow resistance as the CP is being filled (Eddowes, 1988). Such designs have been realised in the form of parallel microchannels arranged in an arborescent pattern (Juncker, 2002) or arrays of posts (Gervais, 2011), or meshes (Mirzaei, 2010). Based on a microfluidic design by Melin et al (Melin, 2004), Safavieh et al reported a CP design with varying capillary flow based on a meandering channel design containing geometric capillary valves between the meander arms.…”

High-throughput tracking of single yeast cells in a microfluidic imaging matrix

“…Combining the scalability and medium exchange capabilities of microfluidics with long-term tracking of specific single cells through time requires methods for the immobilization of cells. Previous approaches for immobilization of non-adherent cells include mechanical clamping under a permeable membrane32, 33, adsorption to channel walls 15, 34, and mechanical trapping by microfabricated constrictions 35-37…”

3D Miniaturization of Human Organs for Drug Discovery

“…For example, cellular components can be integrated by co-culturing multiple cell types in defined physical arrangements, 3D organization can be mimicked with biomaterial scaffolds and microfluidic channels, mechanical cues can be presented by biomaterials and fluid flow, and soluble stimuli can be delivered via perfusion. [35][36][37][38] Top-down approaches include organ-on-a-chip models, which aim to generate key aspects of an organ structure and function in a microfluidic device. Bottom-up approaches rely on the emergent behavior of biological systems to generate complex tissue-and organ-like constructs.…”