; Collagen microsphere production on a chip{ Sungmin Hong, a Hui-Ju Hsu, b Roland Kaunas b and Jun Kameoka* a Received 15th May 2012, Accepted 12th June 2012 DOI: 10.1039/c2lc40558j We have developed an integrated microfluidic material processing chip and demonstrated the rapid production of collagen microspheres encapsulating cells with high uniformity and cell viability. The chip integrated three material processing steps. Monodisperse microdroplets were generated at a microfluidic T junction between aqueous and mineral oil flows. The flow was heated immediately to 37 uC to initiate collagen fiber assembly within a gelation channel. Gelled microspheres were extracted from the mineral oil phase into cell culture media within an extraction chamber. Collagen gelation immediately after microdroplet generation significantly reduced coalescence among microdroplets that led to non-uniform microsphere production. The microfluidic extraction approach led to higher microsphere recovery and cell viability than when a conventional centrifugation extraction approach was employed. These results indicate that chip-based material processing is a promising approach for cell-ECM microenvironment generation for applications such as tissue engineering and stem cell delivery. Introduction Cell-containing microspheres are widely used as building blocks in many biomedical applications such as tissue engineering, 1–4 cell- based biosensors, 5–7 and encapsulated cell delivery. 8–10 These applications require uniform microsphere dimensions and mor- phology with high cell viability. Live cells are typically encapsu- lated in biodegradable polymers such as alginate, 11,12 polyethylene glycol (PEG), 13 and collagen 14,15 that are highly porous, allowing adequate transport of nutrients and oxygen to the cells. Collagen is a particularly attractive material for microspheres as it is the most abundant scaffold protein in tissues 16 and contains specific cell-binding sites that contribute to normal cell function. 17 Collagen microspheres are commonly generated by first forming microdroplets of cells in collagen via direct aliquoting or via emulsification, and subsequently microspheres are formed via gelation of the collagen. Currently, there are no reports regarding the generation of collagen microspheres in microfluidic devices. Aliquoting involves dispensing small volumes (a few microliters) of aqueous collagen solution onto a surface. 9,18–20 In emulsification, aqueous collagen solution is dispersed into microdroplets within a continuous oil phase solution. 21,22 The emulsified microspheres are then typically separated from the oil phase by centrifugation. 23 These conventional approaches are tedious, requiring that each step (i.e. droplet generation, gelation, and extraction) be performed separately. Another drawback of these techniques is the difficulty in maintaining uniform micro- sphere dimensions and shapes at a high production rate. Recently, an axisymmetric flow-focusing device (AFFD) was developed 24 to generate collagen microspheres. The mono-dispersed collagen microdroplets were generated at the orifice of a nozzle in the AFFD and collected in a test tube. The collected collagen microdroplets were gelled at 37 uC for 45 min. Due to the forces generated during centrifugal extraction, the dimensions and shapes of the resulting collagen microspheres were non-uniform due to coalescence of microdroplets. In addition, low cell viability was observed that was attributed to the centrifugation process. 23 A bioprinting platform has also produced cell encapsulated collagen droplets. 25 This approach enables multilayered 3D cell- laden hydrogel structures, high-throughput droplet generation, and long-term viability; however, the shapes of collagen droplets would not be spherical due to gravity. In this paper, an integrated high-throughput microfluidic plat- form was developed to generate collagen microspheres of uniform size and shape with high cell viability. Since this platform integrated the collagen microdroplet generation and the gelation functions on a chip, the time lag between these steps was sufficiently short so as to avoid coalescence of microdroplets, resulting in highly uniform microspheres. Further, a novel microfluidic extraction approach was employed that considerably improved cell viability over that obtained with centrifugal extraction. Experimental Device fabrication Microfluidic devices were fabricated on poly(dimethylsiloxane) substrate (PDMS; Dow Corning, Midland, MI) by standard a Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas, 77843, USA. E-mail: kameoka@mail.ece.tamu.edu b Department of Biomedical Engineering, Texas A&M University, College Station, Texas, 77843, USA { Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c2lc40558j This journal is ß The Royal Society of Chemistry 2012 Lab Chip, 2012, 1–5 | 1 1 5 10 15 20 25 30 35 40 45 50 55 59 1 5 10 15 20 25 30 35 40 45 50 55 59 Lab on a Chip Dynamic Article Links Cite this: DOI: 10.1039/c2lc40558j www.rsc.org/loc TECHNICAL INNOVATION