Abstract—Microdroplet systems can drastically reduce costs and increase throughput in high throughput screening (HTS) assays. While droplets are well suited for biomolecular screening, cell-based screens are more problematic because eukaryotes typically require attachment to solid supports to maintain viability and function. This paper describes an economical, off-the-shelf microfluidic system which encapsulates eukaryotic cells in gelatinous alginate capsules for the purpose of HTS. The flow-through system consists of i) a cross junction, which forms monodisperse droplets of alginate and cell suspension in an immiscible carrier fluid, followed by ii) a T junction which delivers BaCl 2 to crosslink and solidify each droplet. With an appropriate carrier fluid, the system is self-synchronized and can produce cell-alginate-BaCl 2 capsules with virtually 100% reliability. Droplet volumes and frequency are determined by flow rates and the diameter of the cross junction. The present implementation, which utilizes 1.5 mm Teflon tubing and plastic junctions, can generate 0.4-1.4 µL droplets at frequencies >10 droplets/sec. Cell viability is >80% at 4 hours post-encapsulation. With low recurring cost (<$2) and no need for automation robots, this can be an initial step towards economical cell-based HTS. Keywords: droplet, high throughput screening, alginate I. INTRODUCTION igh throughput cell-based assays provide rich information needed in the fields of drug discovery, toxicity testing, genomics, proteomics, and cell biology. Examples include second messenger assays for monitoring signal transduction, reporter assays for monitoring gene expression, and cell viability assays to test for toxicity to external stimuli [1-2]. Current approaches to high throughput screening (HTS) employ microplates with densities ranging from 96-1536 wells/plate, and reaction volumes ranging from 1 mL to 1 µL. The primary concerns with existing technology are the reagent costs and limited throughput. Typical HTS facilities screen 10,000 to 100,000 compounds/day [1] at a cost of $1/assay, determined primarily by the cost of reagents and consumables. Added to this is the initial capital cost of liquid handling robots. Assay throughput is typically < 1 assay/second, limited by reaction rates and the ability of the automation robots to transfer liquids from one container to another. Microdroplet-based HTS has the potential to reduce assay Manuscript received April 23, 2009. V. Trivedi, E. Ereifej, P. Sehgal, and P. VandeVord are with the department of Biomedical Engineering at Wayne State University. A. Doshi and A. Basu are with the department of Electrical and Computer Engineering at Wayne State University, *Correspondence should be addressed to: 5050 Anthony Wayne Drive, Detroit MI, USA. Phone: 313-577-3990; e-mail: abasu@eng.wayne.edu. volumes, increase throughput, and eliminate the need for liquid handling robots, all of which can result in significant cost savings [3]. In microdroplet-based screening, a stream of droplets containing reagents or cells is generated in a microfluidic channel using simple geometries [3-4]. The droplets are separated from each other by an immiscible carrier fluid, allowing each droplet to serve as an isolated reaction container. Droplets can be merged with each other to perform biochemical assays, and inline detectors can provide real time readout of each droplet as they flow by the detector. Microdroplet technology can potentially provide 1) 10-1000x reduction in assay volumes, and 2) throughputs up to 1000 droplets/sec [5-7]. To date, microdroplet systems have been applied primarily towards screening biomolecules. For example, Ismagilov and colleagues used droplet-based screens to optimize conditions for protein crystallization [3,8]. However, there is growing need to perform cell based assays, for the reasons mentioned above. Weitz, Toner, and colleagues demonstrated the encapsulation of single cells into aqueous droplets for the purposes of performing cell-based HTS [5]. Encapsulation directly into an aqueous droplet is a suitable approach for many prokaryotic cells; however, eukaryotes typically require a solid support for cell attachment in order to remain viable and maintain normal function. This requirement is one of the factors limiting the adoption of microdroplet systems for high throughput cell screens. To address this issue, this paper presents a microfluidic system for encapsulating cells into hydrogel capsules which can support cell growth and proliferation. The capsule is made by combining solutions of alginate and Ba 2+ , a multivalent ion which acts as a crosslinking agent. VandeVord and colleagues have previously demonstrated that this type of capsule maintains cell viability >80% and also supports induced secretions in Schwann cells [9]. The capsules can be functionalized with a cell-matrix emulator (Matrigel), and can be further optimized to support cells with additional reagents or proteins. The microfluidic system generates alginate droplets containing cells and then solidifies them inline by adding a fixed amount of the crosslinking agent to each droplet. The resulting capsules are monodisperse, and have a fixed distance between them. The capsules are stored inside a flexible, gas-permeable tube which allows for in situ culture and economical transport between screening facilities. Section II describes the system concept, section III outlines the experimental setup, section IV discusses results, and section V concludes. Microfluidic Encapsulation of Cells in Alginate Capsules for High Throughput Screening Varun Trivedi, Evon S. Ereifej, Ankur Doshi, Priyanka Sehgal, Pamela J. VandeVord, and Amar S. Basu*, Member, IEEE H 7037 31st Annual International Conference of the IEEE EMBS Minneapolis, Minnesota, USA, September 2-6, 2009 978-1-4244-3296-7/09/$25.00 ©2009 IEEE Authorized licensed use limited to: Wayne State University. Downloaded on April 14,2010 at 21:34:10 UTC from IEEE Xplore. Restrictions apply.