A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Abstract

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥10 5 ) in milliliter-scale volumes (≥0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 -2,000 cells) in micro-scale volumes (1 -20 μl). The platform consists of a set of fibronectincoated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities. Video LinkThe video component of this article can be found at

A solvent replenishment solution for managing evaporation of biochemical reactions in air-matrix digital microfluidics devices

“…[2][3][4] Complex reaction series can be carried out using DMF alone, or using hybrid systems in which DMF is integrated with channel-based microfluidics. [5][6][7][8] Hybrid systems offer tremendous versatility; in concept, each reaction step can be executed in the microfluidics format that best accommodates it. We have developed hybrid systems in which DMF is primarily used for multiplexed routing of samples and reagents to and from channel-based microfluidic modules that are specialized to carry out all other needed functions; in this approach, DMF serves to integrate channel-based microfluidic modules with mismatched input/output requirements, obviating the need for complex networks of tubing and microvalves.…”

“…Another strategy is to place the air-matrix DMF device in a closed humidified chamber, 19,20 but this often results in unwanted condensation on the DMF surface, difficult and/or limited access to the device, and need for additional laboratory space and infrastructure. In previous work we avoided these issues by transferring reaction droplets from the air-matrix DMF device to microcapillaries, where they can be heated in dedicated off-chip modules without evaporation problems, 7,8 but this complicates design and manufacture of the air-matrix DMF device, introducing the added complications of microcapillary interfaces and coordination with peripheral modules.…”

The Experimental Side of Parameter Estimation

“…Microscale experimental setups will permit for improved single cell experiment by manipulating cells, see e.g [24,28,49]. New developments also permit to downscale experiments like qPCR [57], or improve spatial configuration of cells or an organism as e.g.…”

World-to-Digital-Microfluidic Interface Enabling Extraction and Purification of RNA from Human Whole Blood

“…Moreover, current and future configurations of the system should support complex downstream processing and analysis, carried out by the DMF device itself 5,14,44−47 and/or additional microfluidics-based modules that are fluidically integrated with the DMF device. 13,46,48…”

“…In DMF, discrete nanoliter- to microliter-sized droplets of fluid are manipulated on a planar hydrophobic surface by applying a series of electrical potentials to an array of electrode pads. , DMF has rapidly become popular for chemical, biological, and medical applications, as it allows straightforward control over multiple reagents (no pumps, valves, or tubing required), − facile handling of both solids and liquids (no channels to clog), − and compatibility with even troublesome reagents (e.g., organic solvents, corrosive chemicals) because the hydrophobic surface (typically Teflon-coated) is chemically inert. , However, a continuing challenge for DMF is handling of “real-world” samples, which typically are composed of fluid volumes greater than those easily accommodated by DMF devices, containing analytes of interest at concentrations too dilute to support downstream processing and detection without prior concentration. Our group − and others − have demonstrated that droplets can be dispensed onto DMF devices from large volumes (hundreds of microliters to milliliters) contained in off-device reservoirs; this only partially addresses the challenge, however, because on-device processing of hundreds or thousands of droplets to collect enough analyte for further manipulation is often not a realistic strategy. Off-device concentration prior to introduction into the DMF device can be a good solution for reagents, because generally they are prepared in large batches for use in hundreds or thousands of reactions.…”