(697e) Development of Autonomous Microfluidic Chemotaxis Assay Platform for Tip-Growing Plant Cell Using Cell Elongation Assisted Capillary Driven Flow

Chemotaxis is a phenomenon that cells exhibit directional movement in response to external chemical cues. Chemotactic behavior of various cells and microorganisms such as bacterial cells, endothelial cells, neurons, and Caenorhabditis elegans have been examined on microfluidic platforms which can offer well-controlled concentration gradient of chemoattractants or repellents with multiplexing capabilities.

Typically, microfluidic-based chemotaxis assay is operated by first introducing chemotactic cells to the micro-channels/chambers, and a chemical environment with either a steady or non-steady state concentration gradient is created around the cells upon the addition of reagents to the reservoirs. For most cases, cells migrate in a particular direction under a certain chemical environment, and researchers evaluate their chemotactic response by quantitating the number of cells in a specific location assigned in the micro-channels/chambers. However, such an end-point analysis may not be applicable for some cases where chemotactic cells do not follow its concentration gradient preciously for a long distance.

A pollen tube (PT) can be categorized in such a hard-to-analyze chemotactic cell in vitro. A PT is a tip-growing plant cell germinated from a single pollen grain and responsible for carrying sperm cells to an egg cell for fertilization. PTs elongate a very long distance and eventually reach to their target (egg cell) by sensing multiple guidance cues from their surroundings.

In this work, we have designed a microfluidic channel network which can guide a single PT to an individually separated reaction chamber where a chemoattractant is autonomously released upon the entrance of a PT in the particular zone.

This idea was realized by creating two regions in the device: (1) PT growth chamber filled with a liquid culture medium, and (2) microchannels filled with chemoattractant in agarose gel. Also, these two regions are connected by an empty channel network. While PTs are initially cultured in region (1), some of them randomly elongate through the empty channel and eventually reach to region (2). During this process, a PT drags the liquid culture medium from region (1) with it, and at the same time, the empty channel is instantly filled with the liquid medium by capillary driven flow. As soon as the liquid reach to region (2), the chemoattractant which has been compartment by air (empty channel) start diffusing in this region, creating a concentration gradient at the tip of a PT. In this situation, when the PT responds to the attractant, it changes its growth direction toward the microchannel filled with the chemical. Therefore, the evaluation of the PT’s chemotactic response can be made by the presence of a PT in the particular channel. As a proof of concept, this chemotactic assay device was demonstrated using Torenia fournieri and its PT attractant peptide, LURE.