Complex Three-Dimensional Fluidic Reservoirs to Control Beads/Living Cells Contacts

Abstract

In this paper, we combine holographic multiple optical tweezers with a three-dimensional microfluidic system to create a versatile microlaboratory. In order to determine cells local and/or temporal response to stimuli, and therefore draw their map of sensitivity, one convenient way is to apply antigen-covered latex beads in order to bind to plasma membrane, by means of optical tweezers. Using multiple optical traps could improve the efficiency of the measurements, but also their versatility. Therefore, we have developed a complete system based on holographic optical tweezers to realise multiple-point interactions between beads and cells with control of the stimulation places, timing, and durations. As we plan to use our system to study biological events in the hour timescale, we have to keep beads and cells separated, in order to prevent unwanted beads to circulate freely in the sample and bind to the target cell during the experiment. We then introduced microstereolithography as a 3D micro-manufacturing approach to the rapid prototyping of three-dimensional fluidic microchambers of complex shapes inside the sample, comprising wells, channels and walls to inject beads locally and keep them separated from cells in our assays. We demonstrated the possibility for microSL to easily and rapidly (typically one hour) fabricate small and three-dimensional observation chambers with customized design of the flow channels, including fluidic reservoirs of typically 500–1500 μm diameter, 5–12 mm height, in order to facilitate manual filling. Several shapes of reservoirs designed to keep beads and cells separated in liquid samples have been realized and successfully tested. Some of them included up to 3 reservoirs, in order to allow co-distribution of different types of beads. Each reservoir typically contained 2–10 μl of solution holding the beads, with a horizontal outlet of 100–200 μm in diameter which allows beads to deposit locally on the microscope cover glass placed under the reservoir outlet. Limited extension of beads under the outlet on the glass has been confirmed, and the ability of the polymeric structures to confine beads in a restricted area has been demonstrated. In the following we present examples of manipulations by multiple holographic optical tweezers consisting at first in extracting several beads from such an area by going through an aperture designed in the structure, making them travel to the target cell, and finally depositing on its outer membrane.